2&#39;-C-methyl-3&#39;-O-L-valine ester ribofuranosyl cytidine for treatment of flaviviridae infections

ABSTRACT

The 3′-L-valine ester of β-D-2′-C-methyl-ribofuranosyl cytidine provides superior results against flaviviruses and pestiviruses, including hepatitis C virus. Based on this discovery, compounds, compositions, methods and uses are provided for the treatment of flaviviridae, including HCV, that include the administration of an effective amount of val-mCyd or its salt, ester, prodrug or derivative, optionally in a pharmaceutically acceptable carrier. In an alternative embodiment, val-mCyd is used to treat any virus that replicates through an RNA-dependent RNA polymerase.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S.Provisional application No. 60/392,351, filed Jun. 28, 2002; U.S.Provisional Application No. 60/466,194, filed Apr. 28, 2003; and U.S.Provisional application No. 60/470,949 entitled “Nucleosides for theTreatment of Infection by Coronaviruses, Togaviruses and Picornaviruses”filed May 14, 2003, the disclosures of each of which are incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] This invention is in the area of pharmaceutical chemistry and, inparticular, is 2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine esterand pharmaceutically acceptable salts, derivatives and prodrugs thereof,their syntheses and their uses as anti-Flaviviridae agents in thetreatment of hosts, notably humans, infected with a Flaviviridae, and inparticular, hepatitis C, virus.

BACKGROUND OF THE INVENTION

[0003] Flaviviridae Viruses

[0004] The Flaviviridae family of viruses comprises at least threedistinct genera: pestiviruses, which cause disease in cattle and pigs;flaviviruses, which are the primary cause of diseases such as denguefever and yellow fever; and hepaciviruses, whose sole member is HCV. Theflavivirus genus includes more than 68 members separated into groups onthe basis of serological relatedness (Calisher et al., J. Gen. Virol,1993, 70, 37-43). Clinical symptoms vary and include fever, encephalitisand hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe,D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia,Pa., 1996, Chapter 31, 931-959). Flaviviruses of global concern that areassociated with human disease include the dengue hemorrhagic feverviruses (DHF), yellow fever virus, shock syndrome and Japaneseencephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6,251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., NewEng. J Med., 1988, 319, 641-643).

[0005] The pestivirus genus includes bovine viral diarrhea virus (BVDV),classical swine fever virus (CSFV, also called hog cholera virus) andborder disease virus (BDV) of sheep (Moennig, V. et al. Adv. Vir. Res.1992, 41, 53-98). Pestivirus infections of domesticated livestock(cattle, pigs and sheep) cause significant economic losses worldwide.BVDV causes mucosal disease in cattle and is of significant economicimportance to the livestock industry (Meyers, G. and Thiel, H.-J.,Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al, Adv.Vir. Res. 1992, 41, 53-98). Human pestiviruses have not been asextensively characterized as the animal pestiviruses. However,serological surveys indicate considerable pestivirus exposure in humans.

[0006] Pestiviruses and hepaciviruses are closely related virus groupswithin the Flaviviridae family. Other closely related viruses in thisfamily include the GB virus A, GB virus A-like agents, GB virus-B and GBvirus-C (also called hepatitis G virus, HGV). The hepacivirus group(hepatitis C virus; HCV) consists of a number of closely related butgenotypically distinguishable viruses that infect humans. There areapproximately 6 HCV genotypes and more than 50 subtypes. Due to thesimilarities between pestiviruses and hepaciviruses, combined with thepoor ability of hepaciviruses to grow efficiently in cell culture,bovine viral diarrhea virus (BVDV) is often used as a surrogate to studythe HCV virus.

[0007] The genetic organization of pestiviruses and hepaciviruses isvery similar.

[0008] These positive stranded RNA viruses possess a single large openreading frame (ORF) encoding all the viral proteins necessary for virusreplication. These proteins are expressed as a polyprotein that is co-and post-translationally processed by both cellular and virus-encodedproteinases to yield the mature viral proteins. The viral proteinsresponsible for the replication of the viral genome RNA are locatedwithin approximately the carboxy-terminal. Two-thirds of the ORF aretermed nonstructural (NS) proteins. The genetic organization andpolyprotein processing of the nonstructural protein portion of the ORFfor pestiviruses and hepaciviruses is very similar. For both thepestiviruses and hepaciviruses, the mature nonstructural (NS) proteins,in sequential order from the amino-terminus of the nonstructural proteincoding region to the carboxy-terminus of the ORF, consist of p7, NS2,NS3, NS4A, NS4B, NS5A, and NS5B.

[0009] The NS proteins of pestiviruses and hepaciviruses share sequencedomains that are characteristic of specific protein functions. Forexample, the NS3 proteins of viruses in both groups possess amino acidsequence motifs characteristic of serine proteinases and of helicases(Gorbalenya et al. (1988) Nature 333:22; Bazan and Fletterick (1989)Virology 171:637-639; Gorbalenya et al. (1989) Nucleic Acid Res.17.3889-3897). Similarly, the NS5B proteins of pestiviruses andhepaciviruses have the motifs characteristic of RNA-directed RNApolymerases (Koonin, E. V. and Dolja, V. V. (1993) Crit. Rev. Biochem.Molec. Biol. 28:375-430).

[0010] The actual roles and functions of the NS proteins of pestivirusesand hepaciviruses in the lifecycle of the viruses are directlyanalogous. In both cases, the NS3 serine proteinase is responsible forall proteolytic processing of polyprotein precursors downstream of itsposition in the ORF (Wiskerchen and Collett (1991) Virology 184:341-350;Bartenschlager et al. (1993) J. Virol. 67:3835-3844; Eckart et al.(1993) Biochem. Biophys. Res. Comm. 192:399-406; Grakoui et al. (1993)J. Virol. 67:2832-2843; Grakoui et al. (1993) Proc. Natl. Acad. Sci. USA90:10583-10587; Hijikata et al. (1993) J. Virol. 67:4665-4675; Tome etal. (1993) J. Virol. 67:4017-4026). The NS4A protein, in both cases,acts as a cofactor with the NS3 serine protease (Bartenschlager et al.(1994) J. Virol. 68:5045-5055; Failla et al. (1994) J. Virol. 68:3753-3760; Lin et al. (1994) 68:8147-8157; Xu et al. (1997) J. Virol.71:5312-5322). The NS3 protein of both viruses also functions as ahelicase (Kim et al. (1995) Biochem. Biophys. Res. Comm. 215: 160-166;Jin and Peterson (1995) Arch. Biochem. Biophys., 323:47-53; Warrener andCollett (1995) J. Virol. 69:1720-1726). Finally, the NS5B proteins ofpestiviruses and hepaciviruses have the predicted RNA-directed RNApolymerases activity (Behrens et al. (1996) EMBO J. 15:12-22; Lehmannetal. (l 997) J. Virol. 71:8416-8428; Yuan et al. (1997) Biochem. Biophys.Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; Zhong et al. (1998)J. Virol. 72.9365-9369).

[0011] Hepatitis C Virus

[0012] The hepatitis C virus (HCV) is the leading cause of chronic liverdisease worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000). HCVcauses a slow growing viral infection and is the major cause ofcirrhosis and hepatocellular carcinoma (Di Besceglie, A. M. and Bacon,B. R., Scientific American, October: 80-85, (1999); Boyer, N. et al. J.Hepatol. 32:98-112, 2000). An estimated 170 million persons are infectedwith HCV worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000).Cirrhosis caused by chronic hepatitis C infection accounts for8,000-12,000 deaths per year in the United States, and HCV infection isthe leading indication for liver transplantation.

[0013] HCV is known to cause at least 80% of posttransfusion hepatitisand a substantial proportion of sporadic acute hepatitis. Preliminaryevidence also implicates HCV in many cases of “idiopathic” chronichepatitis, “cryptogenic” cirrhosis, and probably hepatocellularcarcinoma unrelated to other hepatitis viruses, such as Hepatitis BVirus (HBV). A small proportion of healthy persons appear to be chronicHCV carriers, varying with geography and other epidemiological factors.

[0014] The numbers may substantially exceed those for HBV, thoughinformation is still preliminary; how many of these persons havesubclinical chronic liver disease is unclear. (The Merck Manual, ch. 69,p. 901, 16th ed., (1992)).

[0015] HCV is an enveloped virus containing a positive-sensesingle-stranded RNA genome of approximately 9.4 kb. The viral genomeconsists of a 5′ untranslated region (UTR), a long open reading frameencoding a polyprotein precursor of approximately 3011 amino acids, anda short 3′ UTR. The 5′ UTR is the most highly conserved part of the HCVgenome and is important for the initiation and control of polyproteintranslation. Translation of the HCV genome is initiated by acap-independent mechanism known as internal ribosome entry. Thismechanism involves the binding of ribosomes to an RNA sequence known asthe internal ribosome entry site (IRES). An RNA pseudoknot structure hasrecently been determined to be an essential structural element of theHCV IRES. Viral structural proteins include a nucleocapsid core protein(C) and two envelope glycoproteins, E1 and E2. HCV also encodes twoproteinases, a zinc-dependent metalloproteinase encoded by the NS2NS3region and a serine proteinase encoded in the NS3 region. Theseproteinases are required for cleavage of specific regions of theprecursor polyprotein into mature peptides. The carboxyl half ofnonstructural protein 5, NS5B, contains the RNA-dependent RNApolymerase. The function of the remaining nonstructural proteins, NS4Aand NS4B, and that of NS5A (the amino-terminal half of nonstructuralprotein 5) remain unknown.

[0016] A significant focus of current antiviral research is directed tothe development of improved methods of treatment of chronic HCVinfections in humans (Di Besceglie, A. M. and Bacon, B. R., ScientificAmerican, October: 80-85, (1999)).

[0017] Treatment of HCV Infection with Interferon

[0018] Interferons (IFNs) have been commercially available for thetreatment of chronic hepatitis for nearly a decade. IFNs areglycoproteins produced by immune cells in response to viral infection.IFNs inhibit replication of a number of viruses, including HCV, and whenused as the sole treatment for hepatitis C infection, IFN can in certaincases suppress serum HCV-RNA to undetectable levels. Additionally, IFNcan normalize serum amino transferase levels. Unfortunately, the effectof IFN is temporary and a sustained response occurs in only 8%-9% ofpatients chronically infected with HCV (Gary L. Davis. Gastroenterology118:S104-S114, 2000). Most patients, however, have difficulty toleratinginterferon treatment, which causes severe flu-like symptoms, weightloss, and lack of energy and stamina.

[0019] A number of patents disclose Flaviviridae, including HCV,treatments, using interferon-based therapies. For example, U.S. Pat. No.5,980,884 to Blatt et al. discloses methods for retreatment of patientsafflicted with HCV using consensus interferon. U.S. Pat. No. 5,942,223to Bazer et al. discloses an anti-HCV therapy using ovine or bovineinterferon-tau. U.S. Pat. No. 5,928,636 to Alber et al. discloses thecombination therapy of interleukin-12 and interferon alpha for thetreatment of infectious diseases including HCV. U.S. Pat. No. 5,849,696to Chretien et al. discloses the use of thymosins, alone or incombination with interferon, for treating HCV. U.S. Pat. No. 5,830,455to Valtuena et al. discloses a combination HCV therapy employinginterferon and a free radical scavenger. U.S. Pat. No. 5,738,845 toImakawa discloses the use of human interferon tau proteins for treatingHCV. Other interferon-based treatments for HCV are disclosed in U.S.Pat. No. 5,676,942 to Testa et al., U.S. Pat. No. 5,372,808 to Blatt etal., and U.S. Pat. No. 5,849,696. A number of patents also disclosepegylated forms of interferon, such as, U.S. Pat. Nos. 5,747,646,5,792,834 and 5,834,594 to Hoffmann-La Roche Inc; PCT Publication No. WO99/32139 and WO 99/32140 to Enzon; WO 95/13090 and U.S. Pat. Nos.5,738,846 and 5,711,944 to Schering; and U.S. Pat. No. 5,908,621 to Glueet al.

[0020] Interferon alpha-2a and interferon alpha-2b are currentlyapproved as monotherapy for the treatment of HCV. ROFERON®-A (Roche) isthe recombinant form of interferon alpha-2a. PEGASYS® (Roche) is thepegylated (i.e. polyethylene glycol modified) form of interferonalpha-2a. INTRON® (Schering Corporation) is the recombinant form ofInterferon alpha-2b, and PEG-INTRON® (Schering Corporation) is thepegylated form of interferon alpha-2b.

[0021] Other forms of interferon alpha, as well as interferon beta,gamma, tau and omega are currently in clinical development for thetreatment of HCV. For example, INFERGEN (interferon alphacon-1) byInterMune, OMNIFERON (natural interferon) by Viragen, ALBUFERON by HumanGenome Sciences, REBIF (interferon beta-1a) by Ares-Serono, OmegaInterferon by BioMedicine, Oral Interferon Alpha by AmarilloBiosciences, and interferon gamma, interferon tau, and interferongamma-1b by InterMune are in development.

[0022] Ribivarin

[0023] Ribavirin (1-β-D-ribofuranosyl-1-1,2,4-triazole-3-carboxamide) isa synthetic, non-interferon-inducing, broad spectrum antiviralnucleoside analog sold under the trade name, Virazole (The Merck Index,11th edition, Editor: Budavari, S., Merck & Co., Inc., Rahway, N.J.,p1304, 1989). U.S. Pat. No. 3,798,209 and RE29,835 disclose and claimribavirin. Ribavirin is structurally similar to guanosine, and has invitro activity against several DNA and RNA viruses includingFlaviviridae (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).

[0024] Ribavirin reduces serum amino transferase levels to normal in 40%of patients, but it does not lower serum levels of HCV-RNA (Gary L.Davis. Gastroenterology 118:S104-S114, 2000). Thus, ribavirin alone isnot effective in reducing viral RNA levels. Additionally, ribavirin hassignificant toxicity and is known to induce anemia.

[0025] Ribavirin is not approved fro monotherapy against HCV. It hasbeen approved in combination with interferon alpha-2a or interferonalpha-2b for the treatment of HCV.

[0026] Combination of Interferon and Ribavirin

[0027] The current standard of care for chronic hepatitis C iscombination therapy with an alpha interferon and ribavirin. Thecombination of interferon and ribavirin for the treatment of HCVinfection has been reported to be effective in the treatment ofinterferon naïve patients (Battaglia, A. M. et al., Ann. Pharmacother.34:487-494, 2000), as well as for treatment of patients whenhistological disease is present (Berenguer, M. et al. Antivir. Ther.3(Suppl. 3):125-136, 1998). Studies have show that more patients withhepatitis C respond to pegylated interferon-alpha/ribavirin combinationtherapy than to combination therapy with unpegylated interferon alpha.However, as with monotherapy, significant side effects develop duringcombination therapy, including hemolysis, flu-like symptoms, anemia, andfatigue. (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).

[0028] Combination therapy with PEG-INTRON® (peginterferon alpha-2b) andREBETOL® (Ribavirin, USP) Capsules is available from ScheringCorporation. REBETOL® (Schering Corporation) has also been approved incombination with INTRON® A (Interferon alpha-2b, recombinant, ScheringCorporation). Roche's PEGASYS® (pegylated interferon alpha-2a) andCOPEGUS® (ribavirin) are also approved for the treatment of HCV.

[0029] PCT Publication Nos. WO 99/59621, WO 00/37110, WO 01/81359, WO02/32414 and WO 03/024461 by Schering Corporation disclose the use ofpegylated interferon alpha and ribavirin combination therapy for thetreatment of HCV. PCT Publication Nos. WO 99/15194, WO 99/64016, and WO00/24355 by Hoffmann-La Roche Inc also disclose the use of pegylatedinterferon alpha and ribavirin combination therapy for the treatment ofHCV.

[0030] Additional Methods to Treat Flaviviridae Infections

[0031] The development of new antiviral agents for flaviviridaeinfections, especially hepatitis C, is currently underway. Specificinhibitors of HCV-derived enzymes such as protease, helicase, andpolymerase inhibitors are being developed. Drugs that inhibit othersteps in HCV replication are also in development, for example, drugsthat block production of HCV antigens from the RNA (IRES inhibitors),drugs that prevent the normal processing of HCV proteins (inhibitors ofglycosylation), drugs that block entry of HCV into cells (by blockingits receptor) and nonspecific cytoprotective agents that block cellinjury caused by the virus infection. Further, molecular approaches arealso being developed to treat hepatitis C, for example, ribozymes, whichare enzymes that break down specific viral RNA molecules, and antisenseoligonucleotides, which are small complementary segments of DNA thatbind to viral RNA and inhibit viral replication, are underinvestigation. A number of HCV treatments are reviewed by Bymock et al.in Antiviral Chemistry &Chemotherapy, 11:2; 79-95 (2000) and DeFrancesco et al. in Antiviral Research, 58: 1-16 (2003).

[0032] Examples of classes of drugs that are being developed to treatFlaviviridae infections include:

[0033] (1) Protease Inhibitors

[0034] Substrate-based NS3 protease inhibitors (Attwood et al.,Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood et al.,Antiviral Chemistry and Chemotherapy 1999, 10, 259-273; Attwood et al.,Preparation and use of amino acid derivatives as anti-viral agents,German Patent Pub. DE 19914474; Tung et al. Inhibitors of serineproteases, particularly hepatitis C virus NS3 protease, PCT WO98/17679), including alphaketoamides and hydrazinoureas, and inhibitorsthat terminate in an electrophile such as a boronic acid or phosphonate(Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO99/07734) are being investigated.

[0035] Non-substrate-based NS3 protease inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,Biochemical and Biophysical Research Communications, 1997, 238, 643-647;Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing a para-phenoxyphenylgroup are also being investigated.

[0036] Sch 68631, a phenanthrenequinone, is an HCV protease inhibitor(Chu M. et al., Tetrahedron Letters 37:7229-7232, 1996). In anotherexample by the same authors, Sch 351633, isolated from the fungusPenicillium griseofulvum, was identified as a protease inhibitor (Chu M.et al., Bioorganic and Medicinal Chemistry Letters 9:1949-1952).Nanomolar potency against the HCV NS3 protease enzyme has been achievedby the design of selective inhibitors based on the macromolecule eglinc. Eglin c, isolated from leech, is a potent inhibitor of several serineproteases such as S. griseus proteases A and B, α-chymotrypsin, chymaseand subtilisin. Qasim M. A. et al., Biochemistry 36:1598-1607, 1997.

[0037] Several U.S. patents disclose protease inhibitors for thetreatment of HCV. For example, U.S. Pat. No. 6,004,933 to Spruce et al.discloses a class of cysteine protease inhibitors for inhibiting HCVendopeptidase 2. U.S. Pat. No. 5,990,276 to Zhang et al. disclosessynthetic inhibitors of hepatitis C virus NS3 protease. The inhibitor isa subsequence of a substrate of the NS3 protease or a substrate of theNS4A cofactor. The use of restriction enzymes to treat HCV is disclosedin U.S. Pat. No. 5,538,865 to Reyes et al. Peptides as NS3 serineprotease inhibitors of HCV are disclosed in WO 02/008251 to CorvasInternational, Inc, and WO 02/08187 and WO 02/008256 to ScheringCorporation. HCV inhibitor tripeptides are disclosed in U.S. Pat. Nos.6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim and WO02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3 serineprotease inhibitors of HCV are disclosed in WO 02/48172 to ScheringCorporation. Imidazoleidinones as NS3 serine protease inhibitors of HCVare disclosed in WO 02/08198 to Schering Corporation and WO 02/48157 toBristol Myers Squibb. WO 98/17679 to Vertex Pharmaceuticals and WO02/48116 to Bristol Myers Squibb also disclose HCV protease inhibitors.

[0038] (2) Thiazolidine derivatives which show relevant inhibition in areverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5Bsubstrate (Sudo K. et al., Antiviral Research, 1996, 32, 9-18),especially compound RD-1-6250, possessing a fused cinnamoyl moietysubstituted with a long alkyl chain, RD4 6205 and RD4 6193;

[0039] (3) Thiazolidines and benzanilides identified in Kakiuchi N. etal. J. EBS Letters 421, 217-220; Takeshita N. et al. AnalyticalBiochemistry, 1997, 247, 242-246;

[0040] (4) A phenan-threnequinone possessing activity against proteasein a SDS-PAGE and autoradiography assay isolated from the fermentationculture broth of Streptomyces sp., Sch 68631 (Chu M. et al., TetrahedronLetters, 1996, 37, 7229-7232), and Sch 351633, isolated from the fungusPenicillium griseofulvum, which demonstrates activity in a scintillationproximity assay (Chu M. et al., Bioorganic and Medicinal ChemistryLetters 9, 1949-1952);

[0041] (5) Helicase inhibitors (Diana G. D. et al., Compounds,compositions and methods for treatment of hepatitis C, U.S. Pat. No.5,633,358; Diana G. D. et al., Piperidine derivatives, pharmaceuticalcompositions thereof and their use in the treatment of hepatitis C, PCTWO 97/36554);

[0042] (6) Nucleotide polymerase inhibitors and gliotoxin (Ferrari R. etal. Journal of Virology, 1999, 73, 1649-1654), and the natural productcerulenin (Lohmann V. et al., Virology, 1998, 249, 108-118);

[0043] (7) Antisense phosphorothioate oligodeoxynucleotides (S-ODN)complementary to sequence stretches in the 5′ non-coding region (NCR) ofthe virus (Alt M. et al., Hepatology, 1995, 22, 707-717), or nucleotides326-348 comprising the 3′ end of the NCR and nucleotides 371-388 locatedin the core coding region of the HCV RNA (Alt M. et al., Archives ofVirology, 1997, 142, 589-599; Galderisi U. et al., Journal of CellularPhysiology, 1999, 181, 251-257);

[0044] (8) Inhibitors of IRES-dependent translation (Ikeda N et al.,Agent for the prevention and treatment of hepatitis C, Japanese PatentPub. JP-08268890; Kai Y. et al. Prevention and treatment of viraldiseases, Japanese Patent Pub. JP-10101591);

[0045] (9) Ribozymes, such as nuclease-resistant ribozymes (Maccjak, D.J. et al., Hepatology 1999, 30, abstract 995) and those disclosed inU.S. Pat. No. 6,043,077 to Barber et al., and U.S. Pat. Nos. 5,869,253and 5,610,054 to Draper et al.; and

[0046] (10) Nucleoside analogs have also been developed for thetreatment of Flaviviridae infections.

[0047] Idenix Pharmaceuticals disclosed the use of branched nucleosidesin the treatment of flaviviruses (including HCV) and pestiviruses inInternational Publication Nos. WO 01/90121 and WO 01/92282.Specifically, a method for the treatment of hepatitis C infection (andflaviviruses and pestiviruses) in humans and other host animals isdisclosed in the Idenix publications that includes administering aneffective amount of a biologically active 1′, 2′,3′ or 4′-branched β-Dor β-L nucleosides or a pharmaceutically acceptable salt or derivativethereof, administered either alone or in combination with anotherantiviral agent, optionally in a pharmaceutically acceptable carrier.

[0048] Other patent applications disclosing the use of certainnucleoside analogs to treat hepatitis C virus include: PCT/CA00/01316(WO 01/32153; filed Nov. 3, 2000) and PCT/CA01/00197 (WO 01/60315; filedFeb. 19, 2001) filed by BioChem Pharma, Inc. (now Shire Biochem, Inc.);PCT/US02/01531 (WO 02/057425; filed Jan. 18, 2002) and PCT/US02/03086(WO 02/057287; filed Jan. 18, 2002) filed by Merck & Co., Inc.,PCT/EP01/09633 (WO 02/18404; published Aug. 21, 2001) filed by Roche,and PCT Publication Nos. WO 01/79246 (filed Apr. 13, 2001), WO 02/32920(filed Oct. 18, 2001) and WO 02/48165 by Pharmasset, Ltd.

[0049] PCT Publication No. WO 99/43691 to Emory University, entitled“2′-Fluoronucleosides” discloses the use of certain 2′-fluoronucleosidesto treat HCV.

[0050] Eldrup et al. (Oral Session V, Hepatitis C Virus, Flaviviridae;16^(th) International Conference on Antiviral Research (Apr. 27, 2003,Savannah, Ga.)) described the structure activity relationship of2′-modified nucleosides for inhibition of HCV.

[0051] Bhat et al. (Oral Session V, Hepatitis C Virus, Flaviviridae,2003 (Oral Session V, Hepatitis C Virus, Flaviviridae; 16^(th)International Conference on Antiviral Research (Apr. 27, 2003, Savannah,Ga.); p A75) described the synthesis and pharmacokinetic properties ofnucleoside analogues as possible inhibitors of HCV RNA replication. Theauthors report that 2′-modified nucleosides demonstrate potentinhibitory activity in cell-based replicon assays.

[0052] Olsen et al. (Oral Session V, Hepatitis C Virus, Flaviviridae;16^(th) International Conference on Antiviral Research (Apr. 27, 2003,Savannah, Ga.) p A76) also described the effects of the 2′-modifiednucleosides on HCV RNA replication.

[0053] (11) Other miscellaneous compounds including1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.),alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin E andother antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al.),squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to Ozeki etal.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 toDiana et al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Dianaet al.), polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wanget al.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan etal.), benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et al.), plantextracts (U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No.5,725,859 to Omer et al., and U.S. Pat. No. 6,056,961), and piperidenes(U.S. Pat. No. 5,830,905 to Diana et al.).

[0054] (12) Other compounds currently in preclinical or clinicaldevelopment for treatment of hepatitis C virus include: Interleukin-10by Schering-Plough, IP-501 by Interneuron, Merimebodib (VX-497) byVertex, AMANTADINE® (Symmetrel) by Endo Labs Solvay, HEPTAZYME® by RPI,IDN-6556 by Idun Pharma., XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR®(Hepatitis C Immune Globulin) by NABI, LEVOVIRIN® by ICN/Ribapharm,VIRAMIDINE® by ICN/Ribapharm, ZADAXIN® (thymosin alpha-1) by Sci Clone,thymosin plus pegylated interferon by Sci Clone, CEPLENE® (histaminedihydrochloride) by Maxim, VX 950/LY 570310 by Vertex/Eli Lilly, ISIS14803 by Isis Pharmaceutical/Elan, IDN-6556 by Idun Pharmaceuticals,Inc., JTK 003 by AKROS Pharma, BILN-2061 by Boehringer Ingelheim,CellCept (mycophenolate mofetil) by Roche, T67, a β-tubulin inhibitor,by Tularik, a therapeutic vaccine directed to E2 by Innogenetics, FK788by Fujisawa Healthcare, Inc., IdB 1016 (Siliphos, oralsilybin-phosphatdylcholine phytosome), RNA replication inhibitors(VP50406) by ViroPharma/Wyeth, therapeutic vaccine by Intercell,therapeutic vaccine by Epimmune/Genencor, IRES inhibitor by Anadys, ANA245 and ANA 246 by Anadys, immunotherapy (Therapore) by Avant, proteaseinhibitor by Corvas/SChering, helicase inhibitor by Vertex, fusioninhibitor by Trimeris, T cell therapy by CellExSys, polymerase inhibitorby Biocryst, targeted RNA chemistry by PTC Therapeutics, Dication byImmtech, Int., protease inhibitor by Agouron, protease inhibitor byChiron/Medivir, antisense therapy by AVI BioPharma, antisense therapy byHybridon, hemopurifier by Aethlon Medical, therapeutic vaccine by Merix,protease inhibitor by Bristol-Myers Squibb/Axys, Chron-VacC, atherapeutic vaccine, by Tripep, Utah 231 B by United Therapeutics,protease, helicase and polymerase inhibitors by Genelabs Technologies,IRES inhibitors by Immusol, R803 by Rigel Pharmaceuticals, INFERGEN®(interferon alphacon-1) by InterMune, OMNIFERON® (natural interferon) byViragen, ALBUFERON® by Human Genome Sciences, REBIF (interferon beta-1a)by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alphaby Amarillo Biosciences, interferon gamma, interferon tau, andInterferon gamma-1b by InterMune.

[0055] Nucleoside prodrugs have been previously described for thetreatment of other forms of hepatitis. WO 01/96353 (filed Jun. 15, 2001)to Idenix Pharmaceuticals, discloses 2′-deoxy-β-L-nucleosides and their3′-prodrugs for the treatment of HBV. U.S. Pat. No. 4,957,924 toBeauchamp discloses various therapeutic esters of acyclovir.

[0056] In light of the fact that HCV infection has reached epidemiclevels worldwide, and has tragic effects on the infected patient, thereremains a strong need to provide new effective pharmaceutical agents totreat hepatitis C that have low toxicity to the host.

[0057] Further, given the rising threat of other flaviviridaeinfections, there remains a strong need to provide new effectivepharmaceutical agents that have low toxicity to the host.

[0058] Therefore, it is an object of the present invention to provide acompound, method and composition for the treatment of a host infectedwith hepatitis C virus.

[0059] It is another object of the present invention to provide a methodand composition generally for the treatment of patients infected withpestiviruses, flaviviruses, or hepaciviruses.

SUMMARY OF THE INVENTION

[0060] It has been discovered that the 3′-L-valine ester ofβ-D-2′-C-methyl-ribofuranosyl cytidine (referred to alternatively belowas val-mCyd) provides superior results against flaviviruses andpestiviruses, including hepatitis C virus. Based on this discovery,compounds, compositions, methods and uses are provided for the treatmentof flaviviridae, including HCV, that include the administration of aneffective amount of val-mCyd or its salt, ester, prodrug or derivative,optionally in a pharmaceutically acceptable carrier. In an alternativeembodiment, val-mCyd is used to treat any virus that replicates throughan RNA-dependent RNA polymerase.

[0061] Therefore, a first embodiment provides a compound of Formula (I),β-D-2′-C-methyl-ribofuranosyl cytidine or a pharmaceutically acceptablesalt thereof, and its uses in medical therapy and in the manufacture ofa medicant to treat hosts, particularly humans, infected with aflavivirus or pestivirus, including HCV.

[0062] The compound val-mCyd is converted to the parent mCyd throughde-esterification in the gastrointestinal mucosa, blood or liver, and isactively transported from the gastrointestinal lumen after oral deliveryinto the bloodstream by an amino acid transporter function in the mucosaof the gastrointestinal tract. This accounts for the increase in oralbioavailability compared to the parent 2′-branched nucleoside that istransported primarily by a nucleoside transporter function. There isalso reduced competition with other nucleosides or nucleoside analogsthat are transported by the nucleoside transporter function and not theamino acid transporter function. As partial de-esterification occursprior to complete absorption, the mono or divaline ester continues to beabsorbed using the amino acid transporter function. Therefore, thesuperior outcome of better absorption, bioavailability, and reducedcompetition with other nucleosides or nucleoside analogs for uptake intothe bloodstream is achieved.

[0063] In hepatitis C infected chimpanzees, val-mCyd reduced the meanHCV RNA level by 0.83 log₁₀ copies/ml (8.3 mg/kg/day) and 1.05 log₁₀copies/ml (16.6 mg/kg/day) in seven days. The chimps exhibited nodrug-related safety issues.

[0064] The parent nucleoside (mCyd) framework can exist as a β-D or β-Lnucleoside. In a preferred embodiment, the pharmaceutically acceptablecompound is administered in a form that is at least 90% of the β-Denantiomer. In another embodiment, val-mCyd is at least 95% of the β-Denantiomer. The valine ester also has enantiomeric forms. In a preferredembodiment, the valine moiety is at least 90% of the L-enantiomer. Inanother embodiment, the valine moiety is at least 95% of theL-enantiomer. In alternative embodiments, the compounds are used asracemic mixtures or as any combination of β-D or β-L parent nucleosideand L or D amino acid.

[0065] In one specific embodiment, the compound of Formula (I) isβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester.HCl salt. Inanother specific embodiment, the compound of Formula (II), theβ-D-2′-C-methyl-ribofuranosyl cytidine dihydrochloride salt, is providedfor administration to a host, particularly a human, infected with aflavivirus or pestivirus infection. In other embodiments, tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate, formate,fumarate, propionate, glycolate, lactate, pyruvate, oxalate, maleate,salicyate, sulfate, sulfonate, nitrate, bicarbonate, hydrobromate,hydrobromide, hydroiodide, carbonate, and phosphoric acid salts areprovided. A particularly preferred embodiment is the mono ordihydrochloride salt.

[0066] In another embodiment, a pharmaceutical composition comprising2′-C-methyl-cytidine-3′-O-L-valine ester-or its pharmaceuticallyacceptable salt, including a mono or di HCl salt, ester, prodrug orderivative thereof together with a pharmaceutically acceptable carrier,excipient or diluent is provided.

[0067] In an alternative embodiment, the 5′-hydroxyl group is replacedwith a 5′-OR, wherein R is phosphate (including monophosphate,diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl(including lower acyl); alkyl (including lower alkyl); sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein; alipid, including a phospholipids; an amino acid; a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R is independently H or phosphate.

[0068] The active compounds of the present invention can be administeredin combination or alternation with another agent that is active againsta flavivirus or pestivirus, including HCV (including any described orreferred to in the Background of the Invention), or other usefulbiological agent. Thus another principal embodiment is a pharmaceuticalcomposition that includes β-D-2′-C-methyl-ribofuranosylcytidine-3′-O-L-valine ester-or a pharmaceutically acceptable salt(including a mono- or di-HCl salt), ester, prodrug or pharmaceuticallyacceptable derivative thereof, together with one or more other effectiveantiviral agents, optionally with a pharmaceutically acceptable carrieror diluent. In another embodiment, a method is provided that includesadministering β-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine esteror a salt, ester, prodrug or derivative thereof, in combination or inalternation with one or more second antiviral agents, optionally with apharmaceutically acceptable carrier or diluent.

[0069] Any second antiviral agent may be selected that imparts thedesired biological effect. Nonlimiting examples include interferon,ribavirin, an interleukin, an NS3 protease inhibitor, a cysteineprotease inhibitor, phenanthrenequinone, a thiazolidine derivative,thiazolidine, benzanilide, a helicase inhibitor, a polymerase inhibitor,a nucleotide analogue, gliotoxin, cerulenin, antisense phosphorothioateoligodeoxynucleotides, inhibitor of IRES-dependent translation, orribozyme. In one particular embodiment, the second antiviral agent isselected from natural interferon, interferon alpha (includinginterferon-alpha-2a and interferon-alpha-2b), interferon beta (includinginterferon beta-1a), omega interferon, interferon gamma (includinginterferon gamma-1b), interferon tau, and consensus interferon. Any ofthese interferons can be stabilized or otherwise modified to improve thetolerance and biological stability or other biological properties. Onecommon modification is pegylation (modification with polyethyleneglycol). In one particular embodiment, the second antiviral agent ispegylated or unpegylated interferon 2-alpha.

[0070] In an alternative embodiment, the active compound is a 3′-aminoacid ester of β-D-2′-C-methyl-ribofuranosyl cytidine, wherein the aminoacid can be natural or synthetic and can be in a D or Lstereoconfiguration. In another embodiment, the active compound is a3′-acyl ester of β-D-2′-C-methyl-ribofuranosyl cytidine.

BRIEF DESCRIPTION OF THE FIGURES

[0071]FIGS. 1a and 1 b are illustration of two processes for thepreparation of β-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valineester dihydrochloride, as described in Example 1.

[0072]FIG. 2 is a photocopy of a gel illustrating the site-specificchain termination of in vitro RNA synthesis byβ-D-2′-C-methyl-ribofuranosyl cytidine triphosphate at specified guanineresidues in RNA templates, as described in Example 9.

[0073]FIG. 3 is a graph of the the titer of bovine viral diarrhea virus(BVDV) over number of passages of BVDV infected MDBK cells, indicatingeradication of a persistent BVDV infection by prolonged treatment withβ-D-2′-C-methyl-ribofuranosyl cytidine (16 uM) as described in Example10. Arrows indicate points at which a portion of cells were withdrawnfrom drug treatment.

[0074]FIGS. 4a and 4 b are graphs of the concentration of bovine viraldiarrhea virus (BVDV) in MDBK cells persistently infected with thevirus, as described in Example 11. These graphs indicate the synergybetween β-D-2′-C-methyl-ribofuranosyl cytidine and interferon alpha 2b(IntronA) in reducing the viral titer. FIG. 4a is a graph of the effectof β-D-2′-C-methyl-ribofuranosyl cytidine and IntronA on BVDV (strainNY-1) titers in persistently infected MDBK cells over time. FIG. 4b is agraph of the effect of β-D-2′-C-methyl-ribofuranosyl cytidine incombination with IntronA on BVDV (strain I-N-dIns) titers inpersistently-infected MDBK cells.

[0075]FIGS. 5a-d illustrate the results of experiments studying thedevelopment of resistance to β-D-2′-C-methyl-ribofuranosyl cytidinetreated MDBK cells, infected with bovine viral diarrhea virus (BVDV), asdescribed in Example 12. FIG. 5a is a graph of a representativeexperiment showing the effect over twenty eight days ofβ-D-2′-C-methyl-ribofuranosyl cytidine or IntronA treatment on BVDV(strain I-N-dIns) titers in persistently infected MDBK cells. FIG. 5b isa photocopy of a dish plated with infected MDBK cells that illustratesthe size of the foci formed by phenotypes of the wild-type BVDV (strainI-N-dIns), versus the β-D-2′-C-methyl-ribofuranosyl cytidine-resistantBVDV (I-N-dIns 107R), indicating that the resistant virus formed muchsmaller foci than the wild-type, I-N-dIns strain. FIG. 5c is a graph ofthe titer of BVDV strains I-N-dIns or I-N-dIns-107R over hourspost-infection in infected MDBK cells. FIG. 5d is a graph of the effectof Intron A on the BVDV viral titer yield in de novo-infected MDBK cellstreated with IntronA.

[0076]FIG. 6 is a graph of the concentration of hepatitis C virus(Log₁₀) in individual chimpanzees over days of treatment withβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester as describedin Example 13.

[0077]FIG. 7 is a graph of the concentration of hepatitis C virus inindividual chimpanzees over days of treatment withβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester as comparedto baseline, as described in Example 13.

[0078]FIG. 8 is a graph of percent of totalβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester remaining insamples over time after incubation of the drug in human plasma at 4° C.,21° C., and 37° C., as described in Example 14.

[0079]FIG. 9a is a graph showing the relative levels of the di- andtri-phosphate derivatives of β-D-2′-C-methyl-ribofuranosyl cytidine andβ-D-2′-C-methyl-ribofuranosyl uridine (mUrd) after incubation of HepG2cells with 10 μM β-D-2′-C-methyl-ribofuranosyl cytidine over time, asdescribed in Example 14. FIG. 9b is a graph of the decay of thetri-phosphate derivative of β-D-2′-C-methyl-ribofuranosyl cytidine afterincubation of HepG2 cells with 10 μM β-D-2′-C-methyl-ribofuranosylcytidine over time. FIG. 9c is a graph of the concentration of the di-and tri-phosphate derivatives of β-D-2′-C-methyl-ribofuranosyl cytidineand β-D-2′-C-methyl-ribofuranosyl uridine (mUrd) after incubation ofHepG2 cells with 10 μM β-D-2′-C-methyl-ribofuranosyl cytidine atincreasing concentrations of the drug (μM).

[0080]FIG. 10 is a graph of the concentration (ng/ml) ofβ-D-2′-C-methyl-ribofuranosyl cytidine in human serum afteradministration of β-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valineester to patients, as described in Example 17.

[0081]FIG. 11 is a graph of the median change of the titer of hepatitisC virus in human patients after administration ofβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester, as describedin Example 17. The graph indicates change from baseline in Log₁₀ HCV RNAby patient visit.

DETAILED DESCRIPTION OF THE INVENTION

[0082] It has been discovered that the 3′-L-valine ester ofβ-D-2′-C-methyl-ribofuranosyl cytidine (referred to alternatively belowas val-mCyd) provides superior results against flaviviruses andpestiviruses, including hepatitis C virus. Based on this discovery,compounds, compositions, methods and uses are provided for the treatmentof flaviviridae, including HCV, that include the administration of aneffective amount of val-mCyd or its salt, ester, prodrug or derivative,optionally in a pharmaceutically acceptable carrier. In an alternativeembodiment, val-mCyd is used to treat any virus that replicates throughan RNA-dependent RNA polymerase.

[0083] The disclosed compounds or their pharmaceutically acceptablederivatives or salts or pharmaceutically acceptable formulationscontaining these compounds are useful in the prevention and treatment offlaviviridae (including HCV) infections and other related conditionssuch as anti-HCV antibody positive and HCV-positive conditions, andhepatitis C related hepatic cancer (e.g., hepatocellular carcinoma) andhepatic tumors. In addition, these compounds or formulations can be usedprophylactically to prevent or retard the progression of clinicalillness in individuals who are anti-HCV (or more generallyanti-flaviviridae) antibody or HCV-antigen or flaviviridae-antigenpositive, or who have been exposed to HCV or another flaviviridae virus.

[0084] In summary, the present invention includes the followingfeatures:

[0085] (a) 3′-L-valine ester of β-D-2′-C-methyl-ribofuranosyl cytidine,and pharmaceutically acceptable prodrugs, derivatives and salts thereof,including specifically the mono- and di-hydrochloride salts;

[0086] (b) 3′-L-valine ester of β-D-2′-C-methyl-ribofuranosyl cytidine,and pharmaceutically acceptable prodrugs, derivatives and salts thereoffor use in medical therapy, for example for the treatment or prophylaxisof an flaviridae (including HCV) infection;

[0087] (c) use of the 3′-L-valine ester of β-D-2′-C-methyl-ribofuranosylcytidine, and pharmaceutically acceptable prodrugs, derivatives andsalts thereof in the manufacture of a medicament for treatment of aflaviviridae (including HCV) infection;

[0088] (d) pharmaceutical formulations comprising the 3′-L-valine esterof β-D-2′-C-methyl-ribofuranosyl cytidine, and pharmaceuticallyacceptable prodrugs, derivatives and salts thereof together with apharmaceutically acceptable carrier or diluent;

[0089] (e) processes for the preparation of the 3′-L-valine ester ofβ-D-2′-C-methyl-ribofuranosyl cytidine;

[0090] (f) use of the 3′-L-valine ester of β-D-2′-C-methyl-ribofuranosylin the treatment of infections caused by viruses that replicate throughan RNA dependent RNA polymerase; and

[0091] (g) use of the 3′-L-valine ester of β-D-2′-C-methyl-ribofuranosylin the treatment of viral infections by administration in combination oralternation with another antiviral agent.

[0092] In an alternative embodiment, the active compound is a 3′-aminoacid ester of β-D-2′-C-methyl-ribofuranosyl cytidine, wherein the aminoacid can be natural or synthetic and can be in a D or Lstereoconfiguration. In another embodiment, the active compound is a3′-acyl ester of β-D or β-L 2′-C-methyl-ribofuranosyl cytidine. Thecompounds of this invention either possess antiviral activity, or aremetabolized to a compound that exhibits such activity.

[0093] Although not to be bound by theory, in vitro mechanism-of-actionstudies suggest that mCyd is a specific inhibitor of genomic RNAreplication. Moreover, the intracellular 5′-triphosphate moiety,mCyd-TP, appears to directly inhibit the NS5B RNA-dependent-RNApolymerase. Analysis of RNA synthesis in the presence of mCyd-TPsuggests that mCyd-TP acts as a specific chain terminator of viral RNAsynthesis through as yet unidentified mechanisms. Antiviral nucleosidesand nucleoside analogs are generally converted into the activemetabolite, the 5′-triphosphate (TP) derivatives by intracellularkinases. The nucleoside-TPs then exert their antiviral effect byinhibiting the viral polymerase during virus replication. In culturedcells, intracellular phosphorylation converts mCyd predominantly intothe active species, mCyd-triphosphate (mCyd-TP), along with low levelsof mCyd-diphosphate. Additionally, a second TP product,2′-C-methyl-uridine TP (mUrd-TP), is found and is thought to arise viaintracellular deamination of mCyd or mCyd-5′-phosphate species.

[0094] Flaviviruses included within the scope of this invention arediscussed generally in Fields Virology, Editors: Fields, N., Knipe, D.M. and Howley, P. M.; Lippincott-Raven Pulishers, Philadelphia, Pa.;Chapter 31 (1996). Specific flaviviruses include, without limitation:Absettarov; Alfuy; Apoi; Aroa; Bagaza; Banzi; Bououi; Bussuquara;Cacipacore; Carey Island; Dakar bat; Dengue viruses 1, 2, 3 and 4; EdgeHill; Entebbe bat; Gadgets Gully; Hanzalova; Hypr; Ilheus; Israel turkeymeningoencephalitis; Japanese encephalitis; Jugra; Jutiapa; Kadam;Karshi; Kedougou; Kokoera; Koutango; Kumlinge; Kunjin; Kyasanur Forestdisease; Langat; Louping ill; Meaban; Modoc; Montana myotisleukoencephalitis; Murray valley encephalitis; Naranjal; Negishi; Ntaya;Omsk hemorrhagic fever; Phnom-Penh bat; Powassan; Rio Bravo; Rocio;Royal Farm; Russian spring-summer encephalitis; Saboya; St. Louisencephalitis; Sal Vieja; San Perlita; Saumarez Reef; Sepik; Sokuluk;Spondweni; Stratford; Temusu; Tyuleniy; Uganda S, Usutu, Wesselsbron;West Nile; Yaounde; Yellow fever; and Zika.

[0095] Pestiviruses included within the scope of this invention are alsodiscussed generally in Fields Virology (Id.). Specific pestivirusesinclude, without limitation: bovine viral diarrhea virus (“VDV”);classical swine fever virus (“CSFV”) also known as hog cholera virus);and border disease virus (“DV”).

[0096] Definitions

[0097] The term alkyl, as used herein, unless otherwise specified,refers to a saturated straight, branched, or cyclic, primary, secondary,or tertiary hydrocarbon of typically C₁ to C₁₀, and specificallyincludes CF₃, CCl₃, CFCl₂, CF₂Cl, CH₂CF₃, CF₂CF₃, methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The term includes both substituted and unsubstitutedalkyl groups, and particularly includes halogenated alkyl groups, andeven more particularly fluorinated alkyl groups. Non-limiting examplesof moieties with which the alkyl group can be substituted are selectedfrom the group consisting of halogen (fluoro, chloro, bromo or iodo),hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as taught in Greene, et al., Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

[0098] The term lower alkyl, as used herein, and unless otherwisespecified, refers to a C₁ to C₄ saturated straight, branched, or acyclic (for example, cyclopropyl) alkyl group, including bothsubstituted and unsubstituted forms. Unless otherwise specificallystated in this application, when alkyl is a suitable moiety, lower alkylis preferred. Similarly, when alkyl or lower alkyl is a suitable moiety,unsubstituted alkyl or lower alkyl is preferred.

[0099] The term alkylamino or arylamino refers to an amino group thathas one or two alkyl or aryl substituents, respectively.

[0100] The term “protected” as used herein and unless otherwise definedrefers to a group that is added to an oxygen, nitrogen, or phosphorusatom to prevent its further reaction or for other purposes. A widevariety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis.

[0101] The term aryl, as used herein, and unless otherwise specified,refers to phenyl, biphenyl, or naphthyl, and preferably phenyl. The termincludes both substituted and unsubstituted moieties. The aryl group canbe substituted with any desired moiety, including, but not limited to,one or more moieties selected from the group consisting of halogen(fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamino, arylamino,alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, or phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Second Edition, 1991.

[0102] The term alkaryl or alkylaryl refers to an alkyl group with anaryl substituent.

[0103] The term aralkyl or arylalkyl refers to an aryl group with analkyl substituent.

[0104] The term halo, as used herein, includes a specific description ofchloro, bromo, iodo, and fluoro individually.

[0105] The term purine or pyrimidine base includes, but is not limitedto, adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl isC(O)(alkyl, aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine,N⁶-halopurine, N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine,N⁶-hydroxyalkyl purine, N⁶-alkylaminopurine, N⁶-thioalkyl purine,N²-alkylpurines, N²-alkyl-6-thiopurines, thymine, cytosine,5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil, 5-halouracil,including 5-fluorouracil, C⁵-alkylpyrimidines, 5-iodo-pyrimidine,6-iodo-pyrimidine, 2-Br-vinyl-5-pyrimidine, 2-Br-vinyl-6-pyrimidine,C⁵-benzylpyrimidines, C⁵-halopyrimidines, C⁵-vinylpyrimidine,C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine,C⁵-amidopyrimidine, C⁵-cyanopyrimidine, C⁵-nitropyrimidine,C⁵-amino-pyrimidine, N²-alkylpurines, N²-alkyl-6-thiopurines,5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl,pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Purine bases include, butare not limited to, guanine, adenine, hypoxanthine, 2,6-diaminopurine,and 6-chloropurine. Functional oxygen and nitrogen groups on the basecan be protected as necessary or desired. Suitable protecting groups arewell known to those skilled in the art, and include trimethylsilyl,dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl,trityl, alkyl groups, and acyl groups such as acetyl and propionyl,methanesulfonyl, and β-toluenesulfonyl.

[0106] The term acyl or O-linked ester refers to a group of the formulaC(O)R′, wherein R′ is an straight, branched, or cyclic alkyl (includinglower alkyl), carboxylate residue of an amino acid, aryl includingphenyl, heteroaryl, alkaryl, aralkyl including benzyl, alkoxyalkylincluding methoxymethyl, aryloxyalkyl such as phenoxymethyl; orsubstituted alkyl (including lower alkyl), aryl including phenyloptionally substituted with chloro, bromo, fluoro, iodo, C₁ to C₄ alkylor C₁ to C₄ alkoxy, sulfonate esters such as alkyl or aralkyl sulphonylincluding methanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxy-trityl, substituted benzyl, alkaryl, aralkyl includingbenzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such asphenoxymethyl. Aryl groups in the esters optimally comprise a phenylgroup. In nonlimiting embodiments, acyl groups include acetyl,trifluoroacetyl, methylacetyl, cyclopropylacetyl, cyclopropyl-carboxy,propionyl, butyryl, isobutyryl, hexanoyl, heptanoyloctanoyl,neo-heptanoyl, phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl,2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl,chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl,tert-butylacetyl, trichloroacetyl, monochloroacetyl, dichloroacetyl,7H-dodecafluoro-heptanoyl, perfluoro-heptanoyl, 7H-dodeca20fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl,7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl,7H-dodeca-fluoroheptanoyl, nona-fluoro-3,6-dioxaheptanoyl,nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl,methyl 3-amino-5-phenylthiophene-2-carboxyl,3,6-dichloro-2-methoxy-benzoyl, 4(1,1,2,2-tetrafluoro-ethoxy)-benzoyl,2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl,stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl,perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolylcarbonyl, perfluorocyclohexyl carboxyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,1-pyrrolidinecarbonyl, 4-phenylbenzoyl.

[0107] The term amino acid includes naturally occurring and synthetic α,β, γ, or δ amino acids, and includes but is not limited to, amino acidsfound in proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration, but can also be used in the D-configuration.Alternatively, the amino acid can be a derivative of alanyl, valinyl,leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl,methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl,histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl, β-prolinyl,β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl,β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl,β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl or β-histidinyl.

[0108] As used herein, the term “substantially free of” or“substantially in the absence of” refers to a nucleoside compositionthat includes at least 85 or 90% by weight, preferably 95%, 98, 99% or100% by weight, of the designated enantiomer of that nucleoside.

[0109] Similarly, the term “isolated” refers to a nucleoside compositionthat includes at least 85 or 90% by weight, preferably 95%, 98%, 99% or100% by weight, of the nucleoside.

[0110] The term host, as used herein, refers to an unicellular ormulticellular organism in which the virus can replicate, including celllines and animals, and preferably a human. Alternatively, the host canbe carrying a part of the Flaviviridae viral genome, whose replicationor function can be altered by the compounds of the present invention.The term host specifically refers to infected cells, cells transfectedwith all or part of the Flaviviridae genome and animals, in particular,primates (including chimpanzees) and humans. In most animal applicationsof the present invention, the host is a human patient. Veterinaryapplications, in certain indications, however, are clearly anticipatedby the present invention (such as chimpanzees).

[0111] The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a nucleoside compound which, upon administration to a patient,provides the nucleoside compound. Pharmaceutically acceptable saltsinclude those derived from pharmaceutically acceptable inorganic ororganic bases and acids. Suitable salts include those derived fromalkali metals such as potassium and sodium, alkaline earth metals suchas calcium and magnesium, among numerous other acids well known in thepharmaceutical art. Pharmaceutically acceptable prodrugs refer to acompound that is metabolized, for example hydrolyzed or oxidized, in thehost to form the compound of the present invention. Typical examples ofprodrugs include compounds that have biologically labile protectinggroups on a functional moiety of the active compound. Prodrugs includecompounds that can be oxidized, reduced, aminated, deaminated,hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated,dealkylated, acylated, deacylated, phosphorylated, dephosphorylated toproduce the active compound. The compounds of this invention possessantiviral activity against a Flaviviridae, or are metabolized to acompound that exhibits such activity.

[0112] I. Active Compounds, Physiologically Acceptable Salts andProdrugs Thereof

[0113] Methods and compositions for the treatment of pestivirus,flavivirus and hepatitis C virus infection are described that includeadministering an effective amount of 2′-C-methyl-cytidine-3′-O-L-valine,or a pharmaceutically acceptable salt, ester or prodrug thereof to ahost in need thereof.

[0114] In one embodiment, a hydrochloride salt of the compound ofFormula (I) is used. In another embodiment, a dihydrochloride salt ofthe compound of Formula (I) is preferred. However, the active compoundcan be administered as any salt or prodrug that upon administration tothe recipient is capable of providing directly or indirectly the parentcompound, or that exhibits activity itself. Nonlimiting examples are thepharmaceutically acceptable salts, which are alternatively referred toas “physiologically acceptable salts”, and a compound that has beenalkylated, acylated or otherwise modified at the 5′-position or on thepurine or pyrimidine base, thereby forming a type of “pharmaceuticallyacceptable prodrug”. Further, the modifications can affect thebiological activity of the compound, in some cases increasing theactivity over the parent compound. This can easily be assessed bypreparing the salt or prodrug and testing its antiviral activityaccording to the methods described herein, or other methods known tothose skilled in the art.

[0115] In a first principal embodiment, a compound of the Formula (I),or a pharmaceutically acceptable salt or prodrug thereof, is provided:

[0116] It is to be understood that all stereoisomeric, tautomeric andpolymorphic forms of the compounds are included herein. The 2′-methylsubstitution also may be other alkyl groups such as ethyl, propyl, or,alternatively, ethenyl.

[0117] In an alternative embodiment, the active compound is a 3′-aminoacid ester of β-D-2′-C-methyl-ribofuranosyl cytidine, wherein the aminoacid can be natural or synthetic and can be in a D or Lstereoconfiguration. In another embodiment, the active compound is a3′-acyl ester of β-D-2′-C-methyl-ribofuranosyl cytidine.

[0118] In another alternative embodiment, the 5′-hydroxyl group isreplaced with a 5′-OR, wherein R is phosphate (including monophosphate,diphosphate, triphosphate, or a stabilized phosphate prodrug); astabilized phosphate prodrug; acyl (including lower acyl); alkyl(including lower alkyl); sulfonate ester including alkyl or arylalkylsulfonyl including methanesulfonyl and benzyl, wherein the phenyl groupis optionally substituted with one or more substituents as described inthe definition of aryl given herein; a lipid, including a phospholipids;an amino acid; a carbohydrate; a peptide; cholesterol; or otherpharmaceutically acceptable leaving group which when administered invivo is capable of providing a compound wherein R is independently H orphosphate.

[0119] II. Synthesis of Active Compounds

[0120] The compound of the present invention can be synthesized by meansknown in the art. For example, in one embodiment, a nucleoside,nucleoside analog, or a salt, prodrug, stereoisomer or tautomer thereof,that is disubstituted at the 2° C. is prepared. In another embodiment,β-D-2′-C-methyl-cytidine(4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-C-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one),which has value as an active nucleoside or is used as a processintermediate is prepared. In yet another embodiment, 3′-O-valinyl esterof β-D-2′-C-methyl-cytidine (2-amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-4-C-methyl-2-hydroxymethyl-tetrahydro-furan-3-ylester) or a hydrochloride salt form is prepared. Nucleosides, nucleosideanalogs, salts or ester prodrugs prepared may be used as intermediatesin the preparation of a wide variety of other nucleoside analogues, ormay be used directly as antiviral and/or antineoplastic agents.

EXAMPLE 1 SYNTHESIS OF β-D-2′-C-METHYL-RIBOFURANOSYLCYTIDINE-3′-O-L-VALINE ESTER

[0121] In one synthesis method, depicted in FIG. 1a, the synthesiscomprises reacting cytosine, BSA and SnCl₄/acetonitrile with1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (1) to form4-amino-1-(3,4-dibenzoyloxy-5-benzoyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(2); and reacting (2) with NaOMe/MeOH to provide4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(3), also known as 2-C-methyl-β-D-ribofuranose. The use of cytosine as astarting material rather than benzoyl-cytosine improves the “atomeconomy” of the process and simplifies purification at later steps.

[0122] The next steps in this process comprise reacting (3) withMe₂NCH(OMe)₂ in DMF to form (4),N-[1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl]-N,N-dimethyl-formamidine, which is theamino-protected form of (3); reacting (4) with TBDPSCl and imidazole inDCM to provide the 5′-silyl-protected form of (4) asN′-{1-[5-(tert-butyl-diphenyl-silanyloxymethyl)-3,4-dihydroxy-3-methyl-tetrahydro-furan-2-yl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-N,N-dimethyl-formamidine(5), where the use of DCM provides the advantage of having greatercontrol over disilyl by-product formation; reacting (5) withN-Boc-L-valine, EDC and DMAP in DCM at room temperature to form2-tert-butoxycarbonylamino-3-methyl-butyric acid2-(tert-butyl-diphenyl-silanyloxy-methyl)-5-[4-(dimethylamino-methyleneamino)-2-oxo-2H-pyrimidin-1-yl]-4-hydroxy-4-methyl-tetrahydro-furan-3-ylester (6); removing the silyl and amino-protecting groups by reacting(6) with NH₄F in MeOH in the presence of approximately 10 moleequivalents of ethyl acetate to prevent cleavage of the 3′-O-valinylester by liberated ammonia, and refluxing the mixture to provide2-tert-butoxycarbonylamino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester to provide (2); and finally, reacting (2) with HCl in EtOH toprovide 2-amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydrofuran-3-ylester, dihydrochloride salt (8) as a final product.

[0123] Alternative Synthesis

[0124] In another method to synthesize the compounds of the invention,shown in FIG. 1b, benzoylcytosine, BSA and SnCl₄/acetonitrile arereacted with 1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (1) toform4-benzoylamino-1-(3,4-dibenzoyloxy-5-benzoyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2one(2a); reacting (2a) with NH₃ in methanol and chromatographicallyseparating the product,4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(3), also known as β-D-2′-C-methyl-cytidine; reacting (1 withMe₂NCH(OMe)₂ in DMF at room temperature for 1.5 hours to formN-[1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl]-N,N-dimethyl-formamidine(4); reacting (4) with TBDPSCl and pyridine at room temperature for 6hours to provideN′-{1-[5-(tert-butyl-diphenylsilanyloxymethyl)-3,4-dihydroxy-3-methyl-tetrahydro-furan-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}-N,N-dimethyl-formamidine (5; reacting (1 withN-Boc-L-valine, DEC and DMAP in THF/DMF at room temperature for 2 daysand subjecting the product formed from this reaction to HPLC in order toprovide 2-tert-butoxycarbonylamino-3-methyl-butyric acid2-(tert-butyl-diphenyl-silanyloxy-methyl)-5-[4-(dimethylaminomethyleneamino)-2-oxo-2H-pyrimidin-1-yl]-4-hydroxy-4-methyl-tetrahydro-furan-3-ylester (6); refluxing (6) with NH₄F in MeOH for about 3 hours to removethe silyl and amino-protecting groups, and subjecting the product tochromatographic purification to provide2-tert-butoxycarbonylamino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester (7); and finally reacting (7) with HCl in EtOAc at roomtemperature to provide 2-amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester, dihydrochloride salt (8) as a final product.

EXAMPLE 2 SYNTHESIS OF 2′-C-METHYL-CYTIDINE-3′-O-L-VALINE ESTER(VAL-mCyd)

[0125]

[0126] Step 1: Synthesis of Compound 9: 2-C-Methyl-D-ribonic-γ-lactone

[0127] De-ionized water (100 mL) was stirred in a 250 mL 3-necked roundbottom flask, equipped with an overhead stirrer, a stirring shaft, adigital temperature read-out device and an argon line. Argon was bubbledinto water for thirty minutes and D-fructose (20.0 g, 0.111 mole) wasadded and the solution became clear in a few minutes. Calcium oxide(12.5 g, 0.223 mole) was added in portions over a period of five minutesand the mixture was vigorously stirred. An exotherm was observed andreaction temperature reached 39.6° C. after 10 minutes from the start ofthe calcium oxide addition. After about fifteen minutes, the reactionmixture developed a yellow color that deepened with time. After threehours, an aliquot was withdrawn for TLC analysis. The aliquot wasacidified to pH 2 using saturated aqueous solution of oxalic acid. Theresulting white suspension was evaporated under reduced pressure toremove the water. Toluene (2 mL) was added to the residue and themixture was evaporated under reduced pressure (at 45-50° C.) to removeany trace of water. The residual solid was re-constituted in 2 mL of 1:1tetrahydrofuran:methanol mixture. After thorough mixing, the suspensionwas allowed to settle and the supernatant clear solution was spotted forTLC (silica plate was developed in 2% methanol in ethyl acetate andstained in 1% alkaline potassium permanganate dip. The plate was thenheated, using a heat gun, until the appearance of yellowish spots on thepink background). The desired lactone typically appears at an R_(f)value of 0.33 under the above conditions. More polar by-products andunreacted material are detected in the R_(f) value range of 0.0 to 0.2.

[0128] Although product formation was observed after 3 hours, thereaction was allowed to continue for 22 hours during which time thereaction mixture was stirred at 25° C. At the end of this period, pH ofthe mixture was 13.06. Carbon dioxide gas was bubbled into the reactionmixture for about 2.5 hours (pH was 7.25). The formed calcium carbonatesolid was removed by vacuum filtration, filter cake washed with 50 mL ofde-ionized water. The aqueous layers were combined and treated withoxalic acid (5.0 g, 0.056 mole) and the mixture was vigorously stirredat 25° C. for 30 minutes (The initial dark color largely disappeared andthe mixture turned into a milky white slurry). The pH of the mixture atthis stage is typically 2-3. The slurry mixture was stirred at 45-50° C.overnight. The mixture was then evaporated under reduced pressure and at45-50° C. to remove 75 mL of water. Sodium chloride (30 g) andtetrahydrofuran (100 mL) were added to the aqueous slurry (about 75 mL)and the mixture was vigorously stirred at 25° C. for 30 minutes. Thelayers were separated and the aqueous layer was stirred for 10 minuteswith 75 mL of fresh tetrahydrofuran. This process was repeated for threetimes and the tetrahydrofuran solutions were combined and stirred with10 g of anhydrous magnesium sulfate for 30 minutes. The mixture wasfiltered and the magnesium sulfate filter cake was washed with 60 mL oftetrahydrofuran. The filtrate was evaporated under reduced pressure andat 40° C. to give 10.86 g of crude product as a dark orange semisolid.(For scale up runs tetrahydrofuran will be replaced with acetone insteadof evaporation of crude product to dryness). Crude product was stirredwith acetone (20 mL) at 20° C. for 3 hours. Product was collected byvacuum filtration and the filter cake washed with 12 mL of acetone togive the desired product 9 as white crystalline solid. Product was driedin vacuum to give 2.45 g (13.6% yield). Melting point of compound 9:158-162° C. (literature melting point: 160-161° C.). ¹H NMR (DMSO-d₆) δppm 5.69 (s, 1H, exch. With D₂O), 5.41 (d, 1H, exch. With D₂O), 5.00 (t,1H, exch. With D₂O), 4.15 (m, 1H), 3.73 (m, 2H), 3.52 (m, 1H), 1.22 (s,3H). ¹³C NMR (DMSO-d₆) δ ppm 176.44, 82.95, 72.17, 72.02, 59.63, 20.95.(C₆H₁₀O₅: calcd C, 44.45; H, 6.22. Found: C, 44.34; H, 6.30).

[0129] Step 2: Synthesis of Compound 10:2,3,5-Tri-O-benzoyl-2-C-methyl-D-ribonic-γ-lactone

[0130] A mixture of lactone 1 (3.0 g, 18.50 mmol.),4-dimethylaminopyridine (0.45 g, 3.72 mmol.) and triethylamine (25.27 g,249.72 mmol.) in 1,2-dimethoxy ethane(50 mL) was stirred at 25° C. underargon atmosphere for thirty minutes. This white suspension was cooled to5° C. and benzoyl chloride (11.7 g, 83.23 mmol.) was added over a periodof fifteen minutes. The mixture was stirred at 25° C. for two hours. TLCanalysis (silica, 2% methanol in ethyl acetate) indicated completeconsumption of starting material. Ice cold water (100 g) was added tothe reaction mixture and stirring was continued for thirty minutes. Theformed white solids were collected by vacuum filtration and filter cakewashed with cold water (50 mL). This crude product was stirred withtert-butyl methyl ether (60 mL) at 20° C. for thirty minutes, thenfiltered, filter cake washed with tert-butyl methyl ether (25 mL) anddried in vacuum to give 7.33 g (83.4% yield) of compound 10 as a whitesolid in 97.74% purity (HPLC/AUC). Melting point of compound 10:137-140° C. (literature melting point: 141-142° C.). ¹H NMR (CDCl₃) δppm 8.04 (d, 2H), 7.92 (d, 2H), 7.73 (d, 2H), 7.59 (t, 1H), 7.45 (m,4H), 7.32 (t, 2H), 7.17 (t, 2H), 5.51 (d, 1H), 5.17 (m, 1H), 4.82-4.66(d of an AB quartet, 2H) 1.95, (s, 3H). ¹³C NMR (CDCl₃) δ ppm 172.87,166.17, 166.08, 165.58, 134.06, 133.91, 133.72, 130.09, 129.85, 129.80,129.37, 128.78, 128.60, 128.49, 127.96, 127.89, 79.67, 75.49, 72.60,63.29, 23.80. TOF MS ES+(M+1: 475).

[0131] Step 3: Synthesis of Compound 11:2,3,5-Tri-O-benzoyl-2-C-methyl-β-D-ribofuranose:

[0132] A solution of Red-Al (65 wt. % in toluene, 2.0 mL, 6.56 mmol.) inanhydrous toluene (2.0 mL) was stirred at 0° C. under argon atmosphere.A solution of anhydrous ethanol (0.38 mL, 6.56 mmol.) in anhydroustoluene (1.6 mL) was added to the toluene solution over a period of fiveminutes. The resulting mixture was stirred at 0° C. for fifteen minutesand 2 mL (2.18 mmol.) of this Red-Al/ethanol reagent was added to a cold(−5° C.) solution of 2,3,5-tri-O-benzoyl-2-C-methyl-D-ribonolactone 2(475 mg, 1.0 mmol.) in anhydrous toluene (10 mL) over a period of 10minutes. The reaction mixture was stirred at −5° C. for forty minutes.TLC analysis (silica plates, 35% ethyl acetate in heptane) indicatedcomplete consumption of starting material. HPLC analysis indicated only0.1% of starting material remaining. The reaction was quenched withacetone (0.2 mL), water (15 mL) and 1 N HCl (15 mL) at 0° C. and allowedto warm to room temperature. 1 N HCl (5 mL) was added to dissolve theinorganic salts (pH: 2-3). The mixture was extracted with ethyl acetate(3×25 mL) and the organic solution washed with brine (25 mL), dried(anhydrous sodium sulfate, 10 g) and solvent removed under reducedpressure and at temperature of 40° C. to give the desired product 11 inquantitative yield (480 mg). This material was used as is for thesubsequent step.

[0133] Step 4: Synthesis of compound 12:1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose:

[0134] Benzoyl chloride (283 mg, 2.0 mmol.) was added, over a period offive minutes, to a cold solution (5° C.) of compound 11 (480 mg, 1.0mmol.), 4-dimethylaminopyridine (12.3 mg, 0.1 mmol.) and triethylamine(506 mg, 5.0 mmol.) in anhydrous tetrahydrofuran (5 mL). The reactionmixture was stirred at room temperature and under argon atmosphereovernight. HPLC analysis indicated 0.25% of un-reacted startingmaterial. The reaction was quenched by adding ice-cold water (10 g) andsaturated aqueous solution of sodium bicarbonate. Tetrahydrofuran wasremoved under reduced pressure and the mixture was extracted with ethylacetate (50 mL). The organic solution was washed with water (25 mL),brine (25 mL), dried (anhydrous sodium sulfate, 12 g) and solventremoved under reduced pressure to give 650 mg of thick oily product.This crude product was stirred with 5 mL of tert-butyl methyl ether for5 minutes and heptane (5 mL) and water (0.1 mL) were added and stirringwas continued for an additional period of two hours at 20° C. Solidswere collected by vacuum filtration and filter caked washed with 1:1heptane:tert-butyl methyl ether solution (6 mL) and tert-butyl methylether (2 mL). Drying the solid in vacuum gave 300 mg (52%) of desiredproduct 12 (98.43% pure by HPLC/AUC) as a white solid that melted at154-156.3° C. (literature melting point: 155-156° C.). ¹H NMR (CDCl₃) δppm 8.13 (m, 4H), 8.07 (d, 2H), 7.89 (d, 2H), 7.63 (m, 3H), 7.48 (m,6H), 7.15 (m, 3H), 7.06 (s, 1H), 5.86 (dd, 1H), 4.79 (m, 1H), 4.70-4.52(d of an AB quartet, 2H), 1.95, (s, 3H). ¹³C NMR (CDCl₃) δ ppm 166.31,165.83, 165.01, 164.77, 134.01, 133.86, 133.70, 133.17, 130.44, 130.13,129.97, 129.81, 129.59, 129.39, 129.07, 128.84, 128.76, 128.37, 98.01,86.87, 78.77, 76.35, 64.05, 17.07. (C₃₄H₂₈O₉: calcd C, 70.34; H, 4.86.Found: C, 70.20; H, 4.95).

[0135] Step 5: Synthesis of Compound 13:4-Amino-1-(3,4-dibenzoyloxy-5-benzyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one

[0136] Cytosine (89 g, 0.80 mol) was suspended in acetonitrile (900 ml)in a 12 L round bottomed flask equipped with a reflux condenser,overhead stirrer and an argon inlet adapter. The suspension was stirredat 20° C. under argon atmosphere and N,O-bis(trimethylsilyl)acetamide(537 ml, 2.2 mol) was added in one portion. The resulting solution washeated to 80° C. and stirred for an additional hour at the sametemperature. 1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (425.0g, 0.73 mol) was suspended in acetonitrile (4000 ml) and added to thereaction mixture. The reaction mixture became clear after a few minutesand the temperature dropped to ca. 50° C. Tin(IV) chloride (154 ml, 1.31mol) was added over a period of 15 minutes and stirring was continued at80°. After one hour, an aliquot of reaction mixture was quenched byadding aqueous sodium bicarbonate solution and extracting the aqueouslayer with ethyl acetate. The ethyl acetate layer was examined by TLC(silica gel, 20% ethyl acetate in heptane, R_(f) for sugar derivative:0.40). TLC analysis indicated the complete consumption of the sugarderivative. Desired product was detected by TLC using 10% methanol indichloromethane (R_(f): 0.37). The reaction was also monitored by HPLC(Method # 2). Reaction mixture was cooled to 20° C. and quenched byadding saturated aqueous sodium bicarbonate solution (3000 ml) over aperiod of 30 minutes (observed an exotherm when added the first fewdrops of the sodium bicarbonate solution). Solid sodium bicarbonate(1350 g) was added in portions to avoid foaming. The mixture was checkedto make sure that its pH is ≧7. Agitation was stopped and layers wereallowed to separate for 20 minutes. The aqueous layer was drained andstirred with ethyl acetate (1500 ml) and the mixture was allowed toseparate (30 minutes). The organic layer was isolated and combined withthe acetonitrile solution. The organic solution was washed with brine(500 ml) and then solvent stripped to a volume of ca. 750 ml. Productcan be used as is in the subsequent reaction. It may also be furtherstripped to white foamy solid, in quantitative yield. Structure ofcompound 10 was confirmed by ¹H NMR analysis.

[0137] Step 6: Synthesis of compound mCyd:4-Amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one

[0138] Sodium methoxide (13.8 g, 0.26 mol) was added to a solution ofcompound 10 (416 g, 0.73 mol) in methanol (2000 ml). The reactionmixture was stirred at room temperature and monitored by TLC (silicagel, 10% methanol in dichloromethane, R_(f) of compound 9: 0.53) and(silica gel, 30% methanol in dichloromethane, R_(f) of compound 11:0.21). Product started to precipitate after 30 minutes and TLC indicatedreaction completion after two hours. The reaction was also monitored byHPLC (Method # 2). Methanol was removed under reduced pressure to avolume of ca. 500 ml chased with ethanol (2×500 ml) to a volume of ca.500 ml. The residual thick slurry was diluted with 750 ml of ethanol andthe mixture was stirred at 20° C. for one hour. Product was collected byfiltration, filter cake washed with ethanol (100 ml) andtert-butyl-methyl ether (100 ml) and dried to give 168 g (90% yield forthe two steps) of product 11 in purity of >97% (HPLC/AUC). Product wasalso analyzed by ¹H and ¹³C NMR.

[0139] Step 7: Synthesis of Compound 14:2-Tert-butoxycarbonylamino-3-methyl-butyric Acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylEster

[0140] A solution of N-(tert-butoxycarbonyl)-L-valine (46.50 g, 214mmol.), carbonyldiimidazole (34.70 g, 214 mmol.), and anhydroustetrahydrofuran (1000 mL) in a 2 L round bottom flask, was stirred at25° C. under argon for 1.5 hours and then at 40-50° C. for 20 minutes.In a separate 5 L 5-necked round bottom flask, equipped with an overheadstirrer, cooling tower, temperature probe, addition funnel, and an argonline was added4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one(50.0 g, 195 mmol.) and anhydrous N,N-dimethylformamide (1000 mL). Thismixture was heated at 100° C. for 20 minutes until all of thepyrimidine-2-one derivative compound went into solution, and thentriethyl amine (500 mL) and 4-dimethylaminopyridine (2.38 g, 19 mmol)were added to the solution. The mixture was next heated at 97° C. for 20minutes and the tetrahydrofuran solution was added slowly through anaddition funnel over a period of 2 hours, maintaining the temperature nolower than 82° C. The reaction mixture was heated at 82° C. for 1 hourand monitored by HPLC (product=68%, SM=11%, and impurity at about 12min=17%, excluding dimethylaminopyridine). The reaction mixture wascooled to room temperature and then triethylamine and tetrahydrofuranwere removed under vacuum at 30° C. The solution was then neutralizedwith acetic acid to a pH of 7.69. N,N-dimethylformamidine was removedunder vacuum at 35° C. and chased with ethyl acetate (2×200 mL). Thecrude product was stirred with ethyl acetate (500 mL) and water (300mL). The two layers were separated and the aqueous layer was extractedwith ethyl acetate (500 mL). The combined organic layers were washedwith an aqueous saturated brine solution (500 mL). Next the organiclayer was extracted with an aqueous solution of malonic acid (4×400 mL,10 wt. %). The organic layer was checked by TLC (silica, 20% methanol indichloromethane) to make sure that all the desired product was removedfrom the organic layer. The acidic aqueous extracts were combined andcooled in an ice bath and neutralized with triethylamine to a pH of 7.40so that the solids fell out of solution. Ethyl acetate then was added tothe aqueous layer. The white solids were collected by vacuum filtration.Drying the obtained solids in vacuum gave 81.08 g of 99.01 pure (HPLC)first crop.

[0141] Step 8: Synthesis of val-mCyd-2-Amino-3-methyl-butyric Acid5-(4-amino-2-oxo-2H-pyrimidine-1-yl)-4-hydroxy-2hydroxy-methyl-4-methyl-tetrahydro-furan-3-yl Ester (DihydrochlorideSalt)

[0142] A solution of compound 14 (21.0 g, 0.046 mol) in ethanol (168 ml)was stirred in a round bottomed flask equipped with an overhead stirrer,temperature probe, argon line and hydrogen chloride gas bubbler.Hydrogen chloride gas (22 g) was bubbled into the clear solution over aperiod of one hour. The reaction temperature was kept below 30° C. usingan ice-water bath. Solid formation started after a few minutes ofintroducing the hydrogen chloride gas. After 4 hours, HPLC (method # 3)showed only 0.8% of starting material. Solids were collected byfiltration and filter cake washed with ethanol (20 ml) and di-ethylether (100 ml). After drying product under vacuum for 16 hours, 19.06 g(96.5%) of val-mCyd was obtained in 97.26% purity (HPLC, method # 3);m.p. 210° C. (brown), 248-250° C. (melted); ¹H NMR (DMSO-d₆) δ ppm 10.0(s, 1H, 1/2NH₂, D₂O exchangeable), 8.9-8.6 (2 br s, 4H, 1/2NH₂, NH₃, D₂Oexchangeable), 8.42 (d, 1H, H-6, J₅₋₆=7.9 Hz), 6.24 (d, 1H, H-5,J₅-6=7.9 Hz), 5.84 (s, 1H, H-1′), 5.12 (d, 1H, H-3′, J_(3′-4′)=8.8 Hz),4.22 (d, 1H, H-4, J_(3′-4′)=8.7 Hz), 4.0-3.9 (m, 1H, CH), 3.8-3.5 (m,2H, H-5′, H-5″), 2.3-2.1 (m, 1H, CH), 1.16 (s, 3H, CH₃), 1.0 (m, 6H,(CH₃)₂CH); FAB>0 (GT) 713 (2M+H)⁺, 449 (M+G+H)⁺, 357 (M+H)⁺, 246 (S)⁺,112 (B+2H)⁺; FAB<0 (GT) 747 (2M+Cl)⁻, 483 (M+G+Cl)⁻, 391 (M+Cl)⁻, 355(M−H)⁻, 116 (Val)⁻, 110 (B)⁻, 35 (Cl).

[0143] Two different HPLC methods were used to analyze the abovecompounds. Both methods use the following reverse phase column:

[0144] Method 1

[0145] 254 nm. 1.00 ml/min flow rate of an acetonitrile/water lineargradient as described below. 20 minute run time. Five-minuteequilibration between runs. TABLE 1 Retention time of key intermediates:Retention Compound Time Compound 10 10.2 min Compound 11  9.4 minCompound 12 12.9 min

[0146] Method 2:

[0147] Identification is determined at 272 nm. The column used is aWaters Novapak® C18, 3.9×150 mm ID, 4 μm particle size, 60 Å pore sizeor equivalent. The chromatographic conditions are as follows: injectionvolume=10 μl, column temperature=25° C., flow rate=1.00 ml/min,ultraviolet detector at 272 nm, run time is 35 minutes. The systemsuitability requirement for the percent relative standard deviation forthe reference standard is not more than 1.0%. TABLE 2a Purity andimpurities are determined at 272 nm: Solvent A - 20 nM Solvent B -triethylammonium Acetonitrile, Time (minutes) acetate buffer HPLC grade.0.00 100.0 0.0 10.00 85.0 15.0 25.00 5.0 95.0 35.0 5.0 95.0

[0148] TABLE 2b Retention times of key intermediates and final drugsubstance: Compound Retention Time (minutes) Compound mCyd 2.7-2.8Compound 14 15.5 val-mCyd 9.1

[0149] Stereochemistry

[0150] It is appreciated that nucleosides of the present invention haveseveral chiral centers and may exist in and be isolated in opticallyactive and racemic forms. Some compounds may exhibit polymorphism. It isto be understood that the present invention encompasses any racemic,optically-active, diastereomeric, polymorphic, or stereoisomeric form,or mixtures thereof, of a compound of the invention, which possess theuseful properties described herein. It being well known in the art howto prepare optically active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase).

[0151] In particular, since the 1′ and 4′ carbons of the nucleoside arechiral, their nonhydrogen substituents (the base and the CHOR groups,respectively) can be either cis (on the same side) or trans (on oppositesides) with respect to the sugar ring system. The four optical isomerstherefore are represented by the following configurations (whenorienting the sugar moiety in a horizontal plane such that the oxygenatom is in the back): cis (with both groups “up”, which corresponds tothe configuration of naturally occurring β-D nucleosides), cis (withboth groups “down”, which is a nonnaturally occurring β-Lconfiguration), trans (with the C2′ substituent “up” and the C4′substituent “down”), and trans (with the C2′ substituent “down” and theC4′ substituent “up”). The “D-nucleosides” are cis nucleosides in anatural configuration and the “L-nucleosides” are cis nucleosides in thenonnaturally occurring configuration.

[0152] Likewise, most amino acids are chiral (designated as L or D,wherein the L enantiomer is the naturally occurring configuration) andcan exist as separate enantiomers.

[0153] Examples of methods to obtain optically active materials areknown in the art, and include at least the following.

[0154] i) physical separation of crystals—a technique wherebymacroscopic crystals of the individual enantiomers are manuallyseparated. This technique can be used if crystals of the separateenantiomers exist, i.e., the material is a conglomerate, and thecrystals are visually distinct;

[0155] ii) simultaneous crystallization—a technique whereby theindividual enantiomers are separately crystallized from a solution ofthe racemate, possible only if the latter is a conglomerate in the solidstate;

[0156] iii) enzymatic resolutions—a technique whereby partial orcomplete separation of a racemate by virtue of differing rates ofreaction for the enantiomers with an enzyme exists;

[0157] iv) enzymatic asymmetric synthesis—a synthetic technique wherebyat least one step of the synthesis uses an enzymatic reaction to obtainan enantiomerically pure or enriched synthetic precursor of the desiredenantiomer;

[0158] v) chemical asymmetric synthesis—a synthetic technique wherebythe desired enantiomer is synthesized from an achiral precursor underconditions that produce asymmetry (i.e., chirality) in the product,which may be achieved using chiral catalysts or chiral auxiliaries;

[0159] vi) diastereomer separations—a technique whereby a racemiccompound is reacted with an enantiomerically pure reagent (the chiralauxiliary) that converts the individual enantiomers to diastereomers.The resulting diastereomers are then separated by chromatography orcrystallization by virtue of their now more distinct structuraldifferences and the chiral auxiliary later removed to obtain the desiredenantiomer;

[0160] vii) first- and second-order asymmetric transformations—atechnique whereby diastereomers from the racemate equilibrate to yield apreponderance in solution of the diastereomer from the desiredenantiomer or where preferential crystallization of the diastereomerfrom the desired enantiomer perturbs the equilibrium such thateventually in principle all the material is converted to the crystallinediastereomer from the desired enantiomer. The desired enantiomer is thenreleased from the diastereomer;

[0161] viii) kinetic resolutions—this technique refers to theachievement of partial or complete resolution of a racemate (or of afurther resolution of a partially resolved compound) by virtue ofunequal reaction rates of the enantiomers with a chiral, non-racemicreagent or catalyst under kinetic conditions;

[0162] ix) enantiospecific synthesis from non-racemic precursors—asynthetic technique whereby the desired enantiomer is obtained fromnon-chiral starting materials and where the stereochemical integrity isnot or is only minimally compromised over the course of the synthesis;

[0163] x) chiral liquid chromatograph—a technique whereby theenantiomers of a racemate are separated in a liquid mobile phase byvirtue of their differing interactions with a stationary phase. Thestationary phase can be made of chiral material or the mobile phase cancontain an additional chiral material to provoke the differinginteractions;

[0164] xi) chiral gas chromatography—a technique whereby the racemate isvolatilized and enantiomers are separated by virtue of their differinginteractions in the gaseous mobile phase with a column containing afixed non-racemic chiral adsorbent phase;

[0165] xii) extraction with chiral solvents—a technique whereby theenantiomers are separated by virtue of preferential dissolution of oneenantiomer into a particular chiral solvent;

[0166] xiii) transport across chiral membranes—a technique whereby aracemate is placed in contact with a thin membrane barrier. The barriertypically separates two miscible fluids, one containing the racemate,and a driving force such as concentration or pressure differentialcauses preferential transport across the membrane barrier. Separationoccurs as a result of the non-racemic chiral nature of the membranewhich allows only one enantiomer of the racemate to pass through.

[0167] III. Pharmaceutical Compositions

[0168] Hosts, including humans, infected with pestivirus, flavivirus,HCV or another organism replicating through a RNA-dependent RNA viralpolymerase, or for treating any other disorder described herein, can betreated by administering to the patient an effective amount of theactive compound or a pharmaceutically acceptable prodrug or salt thereofin the presence of a pharmaceutically acceptable carrier or dilutent.The active materials can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid or solid form.

[0169] A preferred dose of the compound for pestivirus, flavivirus orHCV will be in the range from about 1 to 50 mg/kg, preferably 1 to 20mg/kg, of body weight per day, more generally 0.1 to about 100 mg perkilogram body weight of the recipient per day. Lower doses may bepreferable, for example doses of 0.5-100 mg, 0.5-50 mg, 0.5-10 mg, or0.5-5 mg per kilogram body weight per day. Even lower doses may beuseful, and thus ranges can include from 0.1-0.5 mg per kilogram bodyweight per day. The effective dosage range of the pharmaceuticallyacceptable salts and prodrugs can be calculated based on the weight ofthe parent nucleoside to e delivered. If the salt or prodrug exhibitsactivity in itself, the effective dosage can be estimated as above usingthe weight of the salt or prodrug, or by other means known to thoseskilled in the art.

[0170] The compound is conveniently administered in unit any suitabledosage form, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Anoral dosage of 50-1000 mg is usually convenient, including in one ormultiple dosage forms of 50, 100, 200, 250, 300, 400, 500, 600, 700,800, 900 or 1000 mgs. Lower doses may be preferable, for example from10-100 or 1-50 mg. Also contemplated are doses of 0.1-50 mg, or 0.1-20mg or 0.1-10.0 mg. Furthermore, lower doses may be utilized in the caseof administration by a non-oral route, as, for example, by injection orinhalation.

[0171] Ideally the active ingredient should be administered to achievepeak plasma concentrations of the active compound of from about 0.2 to70 μM, preferably about 1.0 to 10 μM. This may be achieved, for example,by the intravenous injection of a 0.1 to 5% solution of the activeingredient, optionally in saline, or administered as a bolus of theactive ingredient.

[0172] The concentration of active compound in the drug composition willdepend on absorption, inactivation and excretion rates of the drug aswell as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time, with or without otheranti-viral agents.

[0173] A preferred mode of administration of the active compound isoral. Oral compositions will generally include an inert diluent or anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, theactive compound can be incorporated with excipients and used in the formof tablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can e included as part of thecomposition.

[0174] The tablets, pills, capsules, troches and the like can containany of the following ingredients, or compounds of a similar nature: abinder such as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

[0175] The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

[0176] The compound or a pharmaceutically acceptable prodrug or saltthereof can also be mixed with other active materials that do not impairthe desired action, or with materials that supplement the desiredaction, such as antibiotics, antifungals, anti-inflammatories, or otherantivirals, including other nucleoside compounds. Solutions orsuspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

[0177] If administered intravenously, preferred carriers arephysiological saline or phosphate buffered saline (PBS).

[0178] In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems, biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation.

[0179] Liposomal suspensions (including liposome's targeted to infectedcells with monoclonal antibodies to viral antigens) are also preferredas pharmaceutically acceptable carriers. These may be prepared accordingto methods known to those skilled in the art, for example, as describedin U.S. Pat. No. 4,522,811 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s), such as sterol phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol, in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

[0180] IV. Prodrugs and Derivatives

[0181] Salt Formulations

[0182] Administration of the nucleoside as a pharmaceutically acceptablesalt is within the scope of the invention. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids,which provide a physiological acceptable anion. These include, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, andα-glycerophosphate, formate, fumarate, propionate, glycolate, lactate,pyruvate, oxalate, maleate, and salicyate. Suitable inorganic salts mayalso be formed, including, sulfate, nitrate, bicarbonate, hydrobromate,carbonate salts, and phosphoric acid. A particularly preferredembodiment is the mono or dihydrochloride salt.

[0183] Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made. In one embodiment, the saltis a hydrochloride salt of the compound. In a further embodiment, thepharmaceutically acceptable salt is a dihydrochloride salt of thecompound of Formula (I). The compounds of this invention possessantiviral activity against flavivirus, pestivirus or HCV, or aremetabolized to a compound that exhibits such activity.

[0184] Nucleotide Prodrugs

[0185] The nucleosides described herein can be administered as anucleotide prodrug to increase the activity, bioavailability, stabilityor otherwise alter the properties of the nucleoside. A number ofnucleotide prodrug ligands are known. In general, alkylation, acylationor other lipophilic modification of the mono-, di- or triphosphate ofthe nucleoside reduces polarity and allows passage into cells. Examplesof substituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, and alcohols, such as 1,2-diacylglycerol. Many are described inR. Jones and N. Bischoferger, Antiviral Research, 1995, 27:1-17. Any ofthese can be used in combination with the disclosed nucleosides toachieve a desired effect.

[0186] The active nucleoside can also be provided as a 5′-phosphoetherlipid or a 5′-ether lipid. Non-limiting examples are disclosed in thefollowing references, which are incorporated by reference herein:Kucera, L. S., N. Iyer, E. Leake, A. Raen, Modest E. K., D. L. W., andC. Piantadosi. 1990. “Novel membrane-interactive ether lipid analogsthat inhibit infectious HIV-1 production and induce defective virusformation.” AIDS Res. Hum. Retro Viruses. 6:491-501; Piantadosi, C., J.Marasco C. J., S. L. Morris-Natschke, K. L. Meyer, F. Gumus, J. R.Surles, K. S. Ishaq, L. S. Kucera, N. Iyer, C. A. Wallen, S. Piantadosi,and E. J. Modest. 1991. “Synthesis and evaluation of novel ether lipidnucleoside conjugates for anti-HIV activity.” J. Med. Chem.34:1408.1414; Hosteller, K. Y., D. D. Richman, D. A. Carson, L. M.Stuhmiller, G. M. T. van Wijk, and H. van den Bosch., 1992. “Greatlyenhanced inhibition of human immunodeficiency virus type I replicationin CEM and HT4-6C cells by 3′-deoxythymidine diphosphatedimyristoylglycerol, a lipid prodrug of 3-deoxythymidine.” Antimicro.Agents Chemother. 36:2025.2029; Hosetler, K. Y., L. M. Stuhmiller, H.Lenting, H. van den Bosch, and D. D. Richman, 1990. “Synthesis andantiretroviral activity of phospholipid analogs of azidothymidine andother antiviral nucleosides.” J. Biol. Chem. 265:61127.

[0187] Nonlimiting examples of U.S. patents that disclose suitablelipophilic substituents that can be covalently incorporated into thenucleoside, preferably at the 5′-OH position of the nucleoside orlipophilic preparations thereof, include U.S. Pat. Nos. 5,149,794 (Sep.22, 1992, Yatvin et al.); 5,194,654 (Mar. 16, 1993, Hostetler et al.,5,223,263 (Jun. 29, 1993, Hostetler et al.); 5,256,641 (Oct. 26, 1993,Yatvin et al.); 5,411,947 (May 2, 1995, Hostetler et al.); 5,463,092(Oct. 31, 1995, Hostetler et al.); 5,543,389 (Aug. 6, 1996, Yatvin etal.); 5,543,390 (Aug. 6, 1996, Yatvin et al.); 5,543,391 (Aug. 6, 1996,Yatvin et al.); and 5,554,728 (Sep. 10, 1996; Basava et al.), all ofwhich are incorporated herein by reference. Foreign patent applicationsthat disclose lipophilic substituents that can be attached to thenucleosides of the present invention, or lipophilic preparations,include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910,WO 94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.

[0188] V. Combination or Alternation Therapy

[0189] The active compounds of the present invention can be administeredin combination or alternation with another anti-flavivirus or pestivirusagent, or in particular an anti-HCV agent. In combination therapy,effective dosages of two or more agents are administered together,whereas in alternation or sequential-step therapy, an effective dosageof each agent is administered serially or sequentially. The dosagesgiven will depend on absorption, inactivation and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens and schedules should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions. In preferred embodiments, an anti-HCV (oranti-pestivirus or anti-flavivirus) compound that exhibits an EC₅₀ of10-15 μm, or preferably less than 1-5 μM, is desirable.

[0190] It has been recognized that drug-resistant variants offlaviviruses, pestiviruses or HCV can emerge after prolonged treatmentwith an antiviral agent. Drug resistance most typically occurs bymutation of a gene that encodes for an enzyme used in viral replication.The efficacy of a drug against the viral infection can be prolonged,augmented, or restored by administering the compound in combination oralternation with a second, and perhaps third, antiviral compound thatinduces a different mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution or other parametersof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

[0191] Any of the viral treatments described in the Background of theInvention can be used in combination or alternation with the compoundsdescribed in this specification. Nonlimiting examples include:

[0192] (1) Protease Inhibitors

[0193] Examples include substrate-based NS3 protease inhibitors (Attwoodet al., Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood etal., Antiviral Chemistry and Chemotherapy 1999, 10, 259-273; Attwood etal., Preparation and use of amino acid derivatives as anti-viral agents,German Patent Pub. DE 19914474; Tung et al. Inhibitors of serineproteases, particularly hepatitis C virus NS3 protease, PCT WO98/17679), including alphaketoamides and hydrazinoureas, and inhibitorsthat terminate in an electrophile such as a boronic acid or phosphonate(Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO99/07734); Non-substrate-based NS3 protease inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,Biochemical and Biophysical Research Communications, 1997, 238, 643-647;Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing a para-phenoxyphenylgroup; and Sch 68631, a phenanthrenequinone, an HCV protease inhibitor(Chu M. et al., Tetrahedron Letters 37:7229-7232, 1996).

[0194] Sch 351633, isolated from the fungus Penicillium griseofulvum,was identified as a protease inhibitor (Chu M. et al., Bioorganic andMedicinal Chemistry Letters 9:1949-1952). Eglin c, isolated from leech,is a potent inhibitor of several serine proteases such as S. griseusproteases A and B, α-chymotrypsin, chymase and subtilisin. Qasim M. A.et al., Biochemistry 36:1598-1607, 1997.

[0195] U.S. patents disclosing protease inhibitors for the treatment ofHCV include, for example, U.S. Pat. No. 6,004,933 to Spruce et al. whichdiscloses a class of cysteine protease inhibitors for inhibiting HCVendopeptidase 2; U.S. Pat. No. 5,990,276 to Zhang et al. which disclosessynthetic inhibitors of hepatitis C virus NS3 protease; U.S. Pat. No.5,538,865 to Reyes et a; WO 02/008251 to Corvas International, Inc, andWO 02/08187 and WO 02/008256 to Schering Corporation. HCV inhibitortripeptides are disclosed in U.S. Pat. Nos. 6,534,523, 6,410,531, and6,420,380 to Boehringer Ingelheim and WO 02/060926 to Bristol MyersSquibb. Diaryl peptides as NS3 serine protease inhibitors of HCV aredisclosed in WO 02/48172 to Schering Corporation. Imidazolidindiones asNS3 serine protease inhibitors of HCV are disclosed in WO 02/08198 toSchering Corporation and WO 02/48157 to Bristol Myers Squibb. WO98/17679 to Vertex Pharmaceuticals and WO 02/48116 to Bristol MyersSquibb also disclose HCV protease inhibitors.

[0196] (2) Thiazolidine derivatives which show relevant inhibition in areverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5Bsubstrate (Sudo K. et al., Antiviral Research, 1996, 32, 918),especially compound RD-1-6250, possessing a fused cinnamoyl moietysubstituted with a long alkyl chain, RD4 6205 and RD4 6193;

[0197] (3) Thiazolidines and benzanilides identified in Kakiuchi N. etal. J. EBS Letters 421, 217-220; Takeshita N. et al. AnalyticalBiochemistry, 1997, 247, 242-246;

[0198] (4) A phenan-threnequinone possessing activity against proteasein a SDS-PAGE and autoradiography assay isolated from the fermentationculture broth of Streptomyces sp., Sch 68631 (Chu M. et al, TetrahedronLetters, 1996, 37, 7229-7232), and Sch 351633, isolated from the fungusPenicillium griseofulvum, which demonstrates activity in a scintillationproximity assay (Chu M. et al., Bioorganic and Medicinal ChemistryLetters 9, 1949-1952);

[0199] (5) Helicase inhibitors (Diana G. D. et al., Compounds,compositions and methods for treatment of hepatitis C, U.S. Pat. No.5,633,358; Diana G. D. et al., Piperidine derivatives, pharmaceuticalcompositions thereof and their use in the treatment of hepatitis C, PCTWO 97/36554);

[0200] (6) Nucleotide polymerase inhibitors and gliotoxin (Ferrari R. etal Journal of Virology, 1999, 73, 1649-1654), and the natural productcerulenin (Lohmann V. et al., Virology, 1998, 249, 108-118);

[0201] (7) Antisense phosphorothioate oligodeoxynucleotides (S-ODN)complementary to sequence stretches in the 5′ non-coding region (NCR) ofthe virus (Alt M. et al., Hepatology, 1995, 22, 707-717), or nucleotides326-348 comprising the 3′ end of the NCR and nucleotides 371-388 locatedin the core coding region of the HCV RNA (Alt M. et al., Archives ofVirology, 1997, 142, 589-599; Galderisi U. et al., Journal of CellularPhysiology, 1999, 181, 251-257);

[0202] (8) Inhibitors of IRES-dependent translation (Ikeda N et al,Agent for the prevention and treatment of hepatitis C, Japanese PatentPub. JP-08268890; Kai Y. et al Prevention and treatment of viraldiseases, Japanese Patent Pub. JP-10101591);

[0203] (9) Ribozymes, such as nuclease-resistant ribozymes (Maccjak, D.J. et al., Hepatology 1999, 30, abstract 995) and those disclosed inU.S. Pat. No. 6,043,077 to Barber et al, and U.S. Pat. Nos. 5,869,253and 5,610,054 to Draper et al.; and

[0204] (10) Nucleoside analogs have also been developed for thetreatment of Flaviviridae infections.

[0205] (11) Any of the compounds described by Idenix Pharmaceuticals inInternational Publication Nos. WO 01/90121 and WO 01/92282;

[0206] (12) Other patent applications disclosing the use of certainnucleoside analogs to treat hepatitis C virus include: PCT/CA00/01316(WO 01/32153; filed Nov. 3, 2000) and PCT/CA01/00197 (WO 01/60315; filedFeb. 19, 2001) filed by BioChem Pharma, Inc. (now Shire Biochem, Inc.);PCT/US02/01531 (WO 02/057425; filed Jan. 18, 2002) and PCT/US02/03086(WO 02/057287; filed Jan. 18, 2002) filed by Merck & Co., Inc.,PCT/EP01/09633 (WO 02/18404; published Aug. 21, 2001) filed by Roche,and PCT Publication Nos. WO 01/79246 (filed Apr. 13, 2001), WO 02/32920(filed Oct. 18, 2001) and WO 02/48165 by Pharmasset, Ltd.

[0207] (13) PCT Publication No. WO 99/43691 to Emory University,entitled “2′-Fluoronucleosides” discloses the use of certain2′-fluoronucleosides to treat HCV.

[0208] (14) Other miscellaneous compounds including1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.),alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin E andother antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al.),squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to Ozeki etal.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 toDiana et al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Dianaet al.), polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wanget al.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan etal.), benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et al.), plantextracts (U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No.5,725,859 to Omer et al., and U.S. Pat. No. 6,056,961), and piperadines(U.S. Pat. No. 5,830,905 to Diana et al.).

[0209] (15) Any other compound currently in preclinical or clinicaldevelopment for treatment of hepatitis C virus including: Interleukin-10by Schering-Plough, IP-501 by Interneuron, Merimebodib (VX-497) byVertex, AMANTADINE® (Symmetrel) by Endo Labs Solvay, HEPTAZYME® by RPI,IDN-6556 by Idun Pharma., XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR(Hepatitis C Immune Globulin) by NABI, LEVOVIRIN® by ICN/Ribapharm,VIRAMIDINE® by ICN/Ribapharm, ZADAXIN® (thymosin alpha-1) by Sci Clone,thymosin plus pegylated interferon by Sci Clone, CEPLENE® (histaminedihydrochloride) by Maxim, VX 950/LY 570310 by Vertex/Eli Lilly, ISIS14803 by Isis Pharmaceutical/Elan, IDN-6556 by Idun Pharmaceuticals,Inc., JTK 003 by AKROS Pharma, BILN-2061 by Boehringer Ingelheim,CellCept (mycophenolate mofetil) by Roche, T67, a β-tubulin inhibitor,by Tularik, a therapeutic vaccine directed to E2 by Innogenetics, FK788by Fujisawa Healthcare, Inc., IdB 1016 (Siliphos, oralsilybin-phosphatdylcholine phytosome), RNA replication inhibitors(VP50406) by ViroPharma/Wyeth, therapeutic vaccine by Intercell,therapeutic vaccine by Epimmune/Genencor, IRES inhibitor by Anadys, ANA245 and ANA 246 by Anadys, immunotherapy (Therapore) by Avant, proteaseinhibitor by Corvas/SChering, helicase inhibitor by Vertex, fusioninhibitor by Trimeris, T cell therapy by CellExSys, polymerase inhibitorby Biocryst, targeted RNA chemistry by PTC Therapeutics, Dication byImmtech, Int., protease inhibitor by Agouron, protease inhibitor byChiron/Medivir, antisense therapy by AVI BioPharma, antisense therapy byHybridon, hemopurifier by Aethlon Medical, therapeutic vaccine by Merix,protease inhibitor by Bristol-Myers Squibb/Axys, Chron-VacC, atherapeutic vaccine, by Tripep, Utah 231 B by United Therapeutics,protease, helicase and polymerase inhibitors by Genelabs Technologies,IRES inhibitors by Immusol, R803 by Rigel Pharmaceuticals, INFERGEN®(interferon alphacon-1) by InterMune, OMNIFERON® (natural interferon) byViragen, ALBUFERON® by Human Genome Sciences, REBIF (interferon beta-1a)by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alphaby Amarillo Biosciences, interferon gamma, interferon tau, andInterferon gamma-1b by InterMune.

[0210] V. Biological Data

[0211] Cell Culture Systems for Determining Antiviral Activities

[0212] A useful cell-based assay to detect HCV and its inhibitionassesses the levels of replicon RNA from Huh7 cells harboring the HCVreplicon. These cells can be cultivated in standard media, for exampleDMEM medium (high glucose, no pyruvate), supplemented with 10% fetalbovine serum, 1×non-essential amino acids, Pen-Strep-Glu (100units/liter, 100 microgram/liter, and 2.92 mg/liter, respectively), andG418 (C₂₀H₄₀N₄O₁₀; 500 to 1000 microgram/milliliter). Antiviralscreening assays can be done in the same medium without G418. To keepthe cells in the logarithmic growth phase, cells are seeded in 96-wellplates at low density (for example, 1000 cells per well). The testcompound is then added immediately after seeding the cells and they areincubated for 3 to 7 days at 37° C. in an incubator. The medium is thenremoved, and the cells prepared for total RNA extraction (repliconRNA+host RNA). Replicon RNA can then be amplified in a real-time RT-PCR(Q-RT-PCR) protocol, and quantified.

[0213] The observed differences in quantification of replicon RNA areone way to express the antiviral potency of the test compound. In atypical experiment, a comparable amount of replicon is produced in thenegative control and with non-active compounds. This can be concluded ifthe measured threshold-cycle for HCV RT-PCR in both setting isapproximately the same. In such experiments, a way to express theantiviral effectiveness of a compound is to subtract the averagethreshold RT-PCR cycle of the negative control (Ct_(negative)) from thethreshold RT-PCR cycle of the test compound (Ct_(testcompound)). Thisvalue is called ΔCt (ΔCt=Ct_(testcompound)−Ct_(negative)). A ΔCt valueof 3.3 represents a 1-log reduction in replicon production. As apositive control, recombinant interferon alpha-2a (for example,Roferon-A, Hoffmann-Roche, NJ, USA) can be used alongside the testcompound. Furthermore, the compounds can be tested in dilution series(typically at 100, 33, 10, 3 and 1 μM). The ΔCt values for eachconcentration allow the calculation of the 50% effective concentration(EC₅₀).

[0214] The assay described above can be adapted to the other members ofthe Flaviviridae by changing the cell system and the viral pathogen.Methodologies to determine the efficacy of these antiviral compoundsinclude modifications of the standard techniques as described byHolbrook M R et al. Virus Res. 2000, 69, 31; Markland W et al.Antimicrob. Agents. Chemother. 2000, 44, 859; Diamond M S et al., J.Virol. 2000, 74, 7814; Jordan I et al. J. Infect. Dis. 2000, 182, 1214;Sreenivasan V et al. J. Virol. Methods 1993, 45 (1), 1; or Baginski S Get al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (14), 7981 or thereal-time RT-PCR technology. As an example, an HCV replicon system inHuH7 cells (Lohmann V et al. Science, 1999, 285 (5424), 110) ormodifications thereof (Blight et al. 2000) can be used.

[0215] Non-Cell Based Assays Adapted for Detecting HCV

[0216] Nucleic acid amplification technology is now the method of choicefor identification of a large and still growing number of microorganismssuch as Mycobacterium tuberculosis, human immunodeficiency virus (HIV),and hepatitis C virus (HCV) in biological samples. Nucleic acidamplification techniques include the polymerase chain reaction (PCR),ligase chain reaction (LCR), nucleic acid sequence-based amplification(NASBA), strand-displacement amplification (SDA), andtranscription-mediated amplification (TMA). Several FDA-approveddiagnostic products incorporate these molecular diagnostic methods (seeTable below). Nucleic acid amplification technology tests involve notonly amplification, but detection methodologies as well. The promise ofmolecular diagnostics lies in the improvement of itsspecimen-processing, amplification, and target-detection steps, and inthe integration of these steps into an automated format. AmplificationNucleic Acid FDA- Approved Product: Detection Amplification CommercialAssays Method Method Source C. trachomatis, Heterogeneous: PCR Roche N.gonorrhoeae, Colorimetric Diagnostics M. tuberculosis, HIV-1 C.trachomatis, Heterogeneous: LCR Abbott N. gonorrhoeae ChemiluminescenceLaboratories C. trachomatis, Homogeneous: SDA Becton N. gonorrhoeaeFluorescence Dickinson C. trachomatis, Homogeneous: TMA Gen-Probe M.tuberculosis Chemiluminescence (HPA)

[0217] Amplified-Product Detection Schemes

[0218] Amplified-product detection schemes are of two basic types:heterogeneous and homogeneous. Heterogeneous detection is characterizedby a distinct step, such as washing, designed to remove unhybridizedprobes from hybridized probes, whereas in homogeneous detection there isno physical separation step to remove free probe from bound probe.Multiple heterogeneous and homogeneous detection methods exist. Any ofthese heterogeneous or homogeneous assays can be utilized to assess theeffectiveness of the compounds of the present invention versus anRNA-dependent RNA polymerase virus, such as HCV.

[0219] Heterogeneous Detection: Southern blotting, for example, is aheterogeneous detection technique. In Southern blotting, electrophoresisis used to separate amplification products by size and charge. Thesize-fractionated products are transferred to a membrane or filter bydiffusion, vacuuming, or electroblotting. Labeled detection probes arethen hybridized to the membrane-bound targets in solution, the filtersare washed to remove any unhybridized probe, and the hybridized probe onthe membrane is detected by any of a variety of methods.

[0220] Other types of heterogeneous detection are based on specificcapture of the amplification products by means of enzyme-linkedimmunosorbent assays (ELISAs). One method used with PCR involveslabeling one primer with a hapten or a ligand, such as biotin, and,after amplification, capturing it with an antibody- orstreptavidin-coated microplate. The other primer is labeled with areporter such as fluorescein, and detection is achieved by adding anantifluorescein antibody, horseradish peroxidase (HRP) conjugate. Thistype of method is not as specific as using detection probes thathybridize to defined amplification products of interest.

[0221] The LCx probe system (Abbott Laboratories; Abbott Park, Ill.) andthe Amplicor HIV-1 test (Roche Molecular Systems Inc.; Pleasanton,Calif.) are systems that use heterogeneous detection methods. In the LCxsystem, hapten-labeled oligonucleotide probes thermocycle in the ligasechain reaction. Either a capture hapten or a detection hapten iscovalently attached to each of the four primer oligonucleotides. Uponamplification, each amplified product (amplicon) has one capture haptenand one detection hapten. When amplification is complete, the LCx systeminstrument transfers the reaction to a new well where antibody-coatedmicroparticles bind the capture haptens. Each microparticle is thenirreversibly bound to a glass-fiber matrix. A wash step removes from themicroparticle any probe that contains only the detection hapten. The LCxinstrument adds an alkaline phosphatase (AP)-antibody conjugate thatbinds to the detection hapten. A fluorigenic substrate for AP is4-methylumbelliferyl. Dephosphorylation of 4-methylumbelliferyl by APconverts it to 4-methylumbelliferone, which is fluorescent.

[0222] The Amplicor HIV-1 test uses an ELISA format. After amplificationby PCR, the amplicon is chemically denatured. Amplicon-specificoligonucleotide probes capture the denatured strands onto a coatedmicroplate. The operator washes away any unincorporated primers andunhybridized material in a wash step and then adds an avidin-HRPconjugate to each well. The conjugate binds to the biotin-labeledamplicon captured on the plate. The operator then adds3,3′,5,5′-tetramethylbenzidine (TMB), a chromogenic HRP substrate. Whenhydrogen peroxide is present, HRP oxidizes TMB. The signal is determinedcolorimetrically.

[0223] Homogeneous Detection: Because hybridized and nonhybridizeddetection probes are not physically separated in homogeneous detectionsystems, these methods require fewer steps than heterogeneous methodsand thus are less prone to contamination. Among the commerciallyavailable kits that use homogeneous detection of fluorescent andchemiluminescent labels are the TaqMan system (Applied Biosystems;Foster City, Calif.), BDProbeTecET system (Becton Dickinson; FranklinLakes, N.J.), QPCR System 5000 (Perkin-Elmer Corp.; Norwalk, Conn.), andHybridization Protection Assay (Gen-Probe Inc.; San Diego).

[0224] The TaqMan system detects amplicon in real time. The detectionprobe, which hybridizes to a region inside the amplicon, contains adonor fluorophore such as fluoroscein at its 5′ end and a quenchermoiety, for example, rhodamine, at its 3′ end. When both quencher andfluorophore are on the same oligonucleotide, donor fluorescence isinhibited. During amplification the probe is bound to the target. Taqpolymerase displaces and cleaves the detection probe as it synthesizesthe replacement strand. Cleavage of the detection probe results inseparation of the fluorophore from the quencher, leading to an increasein the donor fluorescence signal. During each cycle of amplification theprocess is repeated. The amount of fluorescent signal increases as theamount of amplicon increases.

[0225] Molecular beacons use quenchers and fluorophores also. Beaconsare probes that are complementary to the target amplicon, but containshort stretches (approximately 5 nucleotides) of complementaryoligonucleotides at each end. The 5′ and 3′ ends of the beacons arelabeled with a fluorophore and a quencher, respectively. A hairpinstructure is formed when the beacon is not hybridized to a target,bringing into contact the fluorophore and the quencher and resulting influorescent quenching. The loop region contains the region complementaryto the amplicon. Upon hybridization to a target, the hairpin structureopens and the quencher and fluorophore separate, allowing development ofa fluorescent signal.¹⁴ A fluorometer measures the signal in real time.

[0226] The BDProbeTecET system uses a real-time detection method thatcombines aspects of TaqMan and molecular beacons. The probe has ahairpin loop structure and contains fluorescein and rhodamine labels. Inthis system, however, the region complementary to the target molecule isnot within the loop but rather in the region 3′ to the rhodamine label.Instead of containing the sequence complementary to the target, thesingle-stranded loop contains a restriction site for the restrictionenzyme BsoBI. The single-stranded sequence is not a substrate for theenzyme. The fluorescein and rhodamine labels are near each other beforeamplification, which quenches the fluorescein fluorescence.Strand-displacement amplification converts the probe into adouble-stranded molecule. The BsoBI restriction enzyme can then cleavethe molecule, resulting in separation of the labels and an increase inthe fluorescent signal.

[0227] The QPCR System 5000 employs electrochemiluminescence withruthenium labels. A biotinylated primer is used. After amplification,the biotin products are captured on streptavidin-coated paramagneticbeads. The beads are transferred into an electrochemical flow cell byaspiration and magnetically held to the surface of the electrode. Uponelectrical stimulation, the ruthenium-labeled probe emits light.

[0228] The Hybridization Protection Assay is used in Gen-Probe'snonamplified PACE assays as well as in amplified Mycobacteriumtuberculosis and Chlamydia trachomatis assays. The detectionoligonucleotide probes in HPA are labeled with chemiluminescentacridinium ester (AE) by means of a linker arm. Hybridization takesplace for 15 minutes at 60° C. in the same tube in which theamplification occurred. The selection reagent, a mildly basic bufferedsolution added after hybridization, hydrolyzes the AE on anyunhybridized probe, rendering it nonchemiluminescent. The AE onhybridized probes folds inside the minor groove of the double helix,thereby protecting itself from hydrolysis by the selection reagent. TheAE emits a chemiluminescent signal upon hydrolysis by hydrogen peroxidefollowed by sodium hydroxide. A luminometer records the chemiluminescentsignal for 2 seconds (a period termed a light-off) and reports thephotons emitted in terms of relative light units (RLU).

[0229] Detection-probe design is critical in all methodologies that useprobes to detect amplification products. Good detection probes hybridizeonly to specified amplification product and do not hybridize tononspecific products. Other key issues in optimizing detectionmethodologies involve the labeling of probes and the maximization ofsample throughput.

[0230] Labeling Methods and Reporter Molecules. Detection probes can belabeled several different ways. Enzymatic incorporation of ³²P or ³⁵Sinto the probes is the most common method for isotopic labeling.Following hybridization and washing, the signal is detected onautoradiographic film.

[0231] To perform nonradioactive detection, probes can be enzymaticallylabeled with a variety of molecules. Biotin can be incorporatedenzymatically and then detected with streptavidin-conjugated alkalinephosphatase, using AP substrates like 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT). Chemiluminescentsubstrates such as Lumi-Phos 530 or Lumi-Phos Plus (Lumigen, Southfield,Mich.) can also be used with AP. In addition, digoxigenin-11-dUTP can beincorporated enzymatically into DNA or RNA, and antidigoxigenin APconjugates can be used with colorimetric or chemiluminescent detection.

[0232] There are numerous other types of reporter molecules, includingchemiluminescent moieties such as acridinium esters. Many fluorescentmoieties are available as well. Electrochemiluminescent compounds suchas tris (2,2′-bipyridine) ruthenium (II) can be used also. Furtherdiscussions of these and similar techniques can be found in: Schiff E R,de Medina M, Kahn R S. Semin Liver Dis. 1999;19(Suppl 1:3-15).

EXAMPLE 3 CELLULAR PHARMACOLOGY OF 2′-C-METHYL-CYTIDINE-3′-O-L-VALINEESTER (VAL-mCyd)

[0233] Phosphorylation Assay of Nucleoside to Active Triphosphate

[0234] To determine the cellular metabolism of the compounds, HepG2cells are obtained from the American Type Culture Collection (Rockville,Md.), and are grown in 225 cm² tissue culture flasks in minimalessential medium supplemented with nonessential amino acids, 1%penicillin-streptomycin. The medium is renewed every three days, and thecells are subcultured once a week. After detachment of the adherentmonolayer with a 10 minute exposure to 30 mL of trypsin-EDTA and threeconsecutive washes with medium, confluent HepG2 cells are seeded at adensity of 2.5×10⁶ cells per well in a 6-well plate and exposed to 10 μMof [³H] labeled active compound (500 dpm/pmol) for the specified timeperiods. The cells are maintained at 37° C. under a 5% CO₂ atmosphere.At the selected time points, the cells are washed three times withice-cold phosphate-buffered saline (PBS). Intracellular active compoundand its respective metabolites are extracted by incubating the cellpellet overnight at −20° C. with 60% methanol followed by extractionwith an additional 20 μL of cold methanol for one hour in an ice bath.The extracts are then combined, dried under gentle filtered air flow andstored at −20° C. until HPLC analysis.

[0235] Antiviral nucleosides and nucleoside analogs are generallyconverted into the active metabolite, the 5′-triphosphate (TP)derivatives by intracellular kinases. The nucleoside-TPs then exerttheir antiviral effect by inhibiting the viral polymerase during virusreplication. In primary human hepatocyte cultures, in a human hepatomacell line (HepG2), and in a bovine kidney cell line (MDBK), mCyd wasconverted into a major metabolite, 2′-C-methyl-cytidine-5′-triphosphate(mCyd-TP), along with smaller amounts of a uridine 5′-triphosphatederivative, 2′-C-methyl-uridine-5′-triphosphate (mUrd-TP). mCyd-TP isinhibitory when tested in vitro against the BVDV replication enzyme, theNS5B RNA dependent RNA polymerase, and was thought to be responsible forthe antiviral activity of mCyd.

[0236] The cellular metabolism of mCyd was examined using MDBK cells,HepG2 cells and human primary hepatocytes exposed to 10 μM [³H]-mCyd.High-pressure liquid chromatography (HPLC) analysis demonstrated thatmCyd was phosphorylated in all three cell types, with mCyd-TP being thepredominant metabolite after 24 h. The metabolic profile obtained over a48-hour exposure of human hepatoma HepG2 cells to 10 μM [³H]-mCyd wastested. In HepG2 cells, levels of mCyd-TP peaked at 41.5±13.4 μM after24 hours (see Table 3) and fell slowly thereafter. In primary humanhepatocytes, the peak mCyd-TP concentration at 24 hours was 4 fold lowerat 10.7±6.7 μM. MDBK bovine kidney cells yielded intermediate levels ofmCyd-TP (30.1±6.9 μM at 24 hours).

[0237] Exposure of hepatocytes to mCyd led to production of a second5′-triphosphate derivative, mUrd-TP. In HepG2 cells exposed to 10 μM[³H]-mCyd, the mUrd-TP level reached 1.9+1.6 μM at 24 hours, compared to8.1±3.4 μM in MDBK cells and 3.2+2.0 μM in primary human hepatocytes.While MDBK and HepG2 cells produced comparable total amounts ofphosphorylated species (approximately 43 versus 47 μM, respectively) at24 h, mUrd-TP comprised 19% of the total product in MDBK cells versusonly 4% in HepG2 cells. mUrd-TP concentration increased steadily overtime, however reached a plateau or declined after 24 hours. TABLE 3Activation of mCyd (10 μM) in Hepatocytes and MDBK Cells Metabolite (μM)Cells^(a) n mCyd-MP mUrd-MP mCyd-DP mUrd-DP mCyd-TP mUrd-TP HepG2 6 NDND  3.7 ± 2.1 ND 41.5 ± 13.4 1.9 ± 1.6 Human 5 ND ND 1.15 ± 1.1 0.26 ±0.4 C 10.7 ± 6.7 3.2 ± 2.0 Primary Hepatocytes MDBK Bovine 7 ND ND  4.2± 2.7 0.76 ± 0.95 30.1 ± 6.9 8.1 ± 3.4 Kidney Cells

[0238] The apparent intracellular half-life of the mCyd-TP was 13.9±2.2hours in HepG2 cells and 7.6±0.6 hours in MDBK cells: the data were notsuitable for calculating the half life of mUrd-TP. Other than thespecific differences noted above, the phosphorylation pattern detectedin primary human hepatocytes was qualitatively similar to that obtainedusing HepG2 or MDBK cells.

EXAMPLE 4 Cell Cytotoxicity

[0239] Mitochondria Toxicity Assay

[0240] HepG2 cells were cultured in 12-well plates as described aboveand exposed to various concentrations of drugs as taught by Pan-ZhouX-R, Cui L, Zhou X-J, Sommadossi J-P, Darley-Usmer V M. “Differentialeffects of antiretroviral nucleoside analogs on mitochondrial functionin HepG2 cells” Antimicrob. Agents Chemother. 2000; 44:496-503. Lacticacid levels in the culture medium after 4 day drug exposure weremeasured using a Boehringer lactic acid assay kit. Lactic acid levelswere normalized by cell number as measured by hemocytometer count.

[0241] Cytotoxicity Assays

[0242] Cells were seeded at a rate of between 5×10³ and 5×10⁴/well into96-well plates in growth medium overnight at 37° C. in a humidified CO₂(5%) atmosphere. New growth medium containing serial dilutions of thedrugs was then added. After incubation for 4 days, cultures were fixedin 50% TCA and stained with sulforhodamineB. The optical density wasread at 550 nm. The cytotoxic concentration was expressed as theconcentration required to reduce the cell number by 50% (CC₅₀).

[0243] Conventional cell proliferation assays were used to assess thecytotoxicity of mCyd and its cellular metabolites in rapidly dividingcells. The inhibitory effect of mCyd was determined to be cytostatic innature since mCyd showed no toxicity in confluent cells atconcentrations far in excess of the corresponding CC₅₀ for a specificcell line. mCyd was not cytotoxic to rapidly growing Huh7 human hepatomacells or H9c2 rat myocardial cells at the highest concentration tested(CC₅₀>250 μM). The mCyd CC₅₀ values were 132 and 161 μM in BHK-21hamster kidney and HepG2 human hepatoma cell lines, respectively. TheCC₅₀ for mCyd in HepG2 cells increased to 200 μM when the cells weregrown on collagen-coated plates for 4 or 10 days. For comparison, CC₅₀values of 35-36 μM were derived when ribavirin was tested in HepG2 andHuh7 cells. In the MDBK bovine kidney cells used for BVDV antiviralstudies, the CC₅₀ of mCyd was 36 μM. A similar CC₅₀ value (34 μM) wasdetermined for mCyd against MT-4 human T-lymphocyte cells. In addition,mCyd was mostly either non-cytotoxic or weakly cytotoxic (CC₅₀>50to >200 μM) to numerous other cell lines of human and other mammalianorigin, including several human carcinoma cell lines, in testingconducted by the National Institutes of Health (NIH) Antiviral Researchand Antimicrobial Chemistry Program. Exceptions to this were rapidlyproliferating HFF human foreskin fibroblasts and MEF mouse embryofibroblasts, where mCyd showed greater cytotoxicity (CC₅₀s 16.9 and 2.4μM, respectively). Again, mCyd was much less toxic to stationary phasefibroblasts.

[0244] The cytotoxic effect of increasing amounts of mCyd on cellularDNA or RNA synthesis was examined in HepG2 cells exposed to[³H]-thymidine or [³H]-uridine. In HepG2 cells, the CC₅₀s of mCydrequired to cause 50% reductions in the incorporation of radiolabeledthymidine and uridine into cellular DNA and RNA, were 112 and 186 μM,respectively. The CC₅₀ values determined for ribavirin (RBV) for DNA andRNA synthesis, respectively, were 3.16 and 6.85 μM. These valuesgenerally reflect the CC₅₀s of 161 and 36 μM determined for mCyd andRBV, respectively, in conventional cell proliferation cytotoxicityassays. To assess the incorporation of mCyd into cellular RNA and DNA,HepG2 cells were exposed to 10 μM [³H]-mCyd or control nucleosides(specific activity 5.6-8.0 Ci/mmole, labeled in the base) for 30 hours.Labeled cellular RNA or DNA species were separately isolated andincorporation was determined by scintillation counting. Exposure ofHepG2 cells to mCyd resulted in very low levels of incorporation of theribonucleoside analog into either cellular DNA or RNA (0.0013-0.0014pmole/μg of nucleic acid). These levels are similar to the 0.0009 and0.0013 pmole/μg values determined for the incorporation of ZDV and ddC,respectively, into RNA: since these deoxynucleosides are not expected toincorporate into RNA, these levels likely reflect the assay background.The incorporation of ZDV and ddC into DNA was significantly higher(0.103 and 0.0055 pmole/1 g, respectively). Ribavirin (RBV) incorporatedinto both DNA and RNA at levels 10-fold higher than mCyd. TABLE 4aCellular Nucleic Acid Synthesis and Incorporation Studies in HepG2 Cells(10 μM Drug and Nucleoside Controls) CC₅₀ (μM) DNA RNA Incorporated drugamount Compound ([³H]Thymidine) ([³H]Uridine) pmole/μg DNA pmole/μg RNAmCyd 112.3 ± 34.5  186.1 ± 28.2  0.0013 ± 0.0008^(a) 0.0014 ± 0.0008^(a)ZDV nd nd  0.103 ± 0.0123^(a) 0.0009 ± 0.0003^(a) ddC nd nd 0.0055^(b)0.0013^(b) Ribavirin 3.16 ± 0.13 6.85 ± 1.83 0.0120^(b) 0.0132^(c)

[0245] TABLE 4b Cytotoxicity of mCyd in Mammalian Cell Lines CellLine^(a) n CC₅₀ (μM) Huh 7 7 >250 Hep G2 6 161 ± 19 Hep G2^(b) 2 >200MDBK 7 36 ± 7 BHK-21 2 132 ± 6  H9c2 2 >250

[0246] Effect on Human Bone Marrow Progenitor Cells

[0247] Bone Marrow Toxicity Assay

[0248] Human bone marrow cells were collected from normal healthyvolunteers and the mononuclear population was separated byFicoll-Hypaque gradient centrifugation as described previously bySommadossi J-P, Carlisle R. “Toxicity of 3′-azido-3′-deoxythymidine and9-(1,3-dihydroxy-2-propoxymethyl)guanine for normal human hematopoieticprogenitor cells in vitro” Antimicrobial Agents and Chemotherapy 1987;31:452-454; and Sommadossi J-P, Schinazi R F, Chu C K, Xie M-Y.“Comparison of cytotoxicity of the (−)- and (+)-enantiomer of2′,3′-dideoxy-3′-thiacytidine in normal human bone marrow progenitorcells” Biochemical Pharmacology 1992; 44:1921-1925. The culture assaysfor CFU-GM and BFU-E were performed using a bilayer soft agar ormethylcellulose method. Drugs were diluted in tissue culture medium andfiltered. After 14 to 18 days at 37° C. in a humidified atmosphere of 5%CO₂ in air, colonies of greater than 50 cells were counted using aninverted microscope. The results are presented as the percent inhibitionof colony formation in the presence of drug compared to solvent controlcultures.

[0249] Cell Protection Assay (CPA)

[0250] The assay was performed essentially as described by Baginski, S.G.; Pevear, D. C.; Seipel, M.; Sun, S. C. C.; Benetatos, C. A.;Chunduru, S. K.; Rice, C. M. and M. S. Collett “Mechanism of action of apestivirus antiviral compound” PNAS USA 2000, 97(14), 7981-7986. MDBKcells (ATCC) were seeded onto 96-well culture plates (4,000 cells perwell) 24 hours before use. After infection with BVDV (strain NADL, ATCC)at a multiplicity of infection (MOI) of 0.02 plaque forming units (PFU)per cell, serial dilutions of test compounds were added to both infectedand uninfected cells in a final concentration of 0.5% DMSO in growthmedium. Each dilution was tested in quadruplicate. Cell densities andvirus inocula were adjusted to ensure continuous cell growth throughoutthe experiment and to achieve more than 90% virus-induced celldestruction in the untreated controls after four days post-infection.After four days, plates were fixed with 50% TCA and stained withsulforhodamine B. The optical density of the wells was read in amicroplate reader at 550 nm. The 50% effective concentration (EC₅₀)values are defined as the compound concentration that achieved 50%reduction of cytopathic effect of the virus.

[0251] The myelosuppressive effects of certain nucleoside analogs havehighlighted the need to test for potential effects of investigationaldrugs on the growth of human bone marrow progenitor cells in clonogenicassays. In particular, anemia and neutropenia are the most commondrug-related clinical toxicities associated with the anti-HIV drugzidovudine (ZDV) or the ribavirin (RBV) component of the standard ofcare combination therapy used for HCV treatment. These toxicities havebeen modeled in an in vitro assay that employed bone marrow cellsobtained from healthy volunteers (Sommadossi J-P, Carlisle R.Antimicrob. Agents Chemother. 1987;31(3): 452-454). ZDV has beenpreviously shown to directly inhibit human granulocyte-macrophagecolony-forming (CFU-GM) and erythroid burst-forming (BFU-E) activity atclinically relevant concentrations of 1-2 μM in this model (Berman E, etal. Blood 1989;74(4):1281-1286; Yoshida Y, Yoshida C. AIDS Res. Hum.Retroviruses 1990;6(7):929-932; Lerza R, et al. Exp. Hematol.1997;25(3):252-255; Dornsife R E, Averett D R. Antimicrob. AgentsChemother. 1996;40(2):514-519; Kurtzberg J, Carter S G. Exp. Hematol.1990;18(10):1094-1096; Weinberg R S, et al. Mt. Sinai. J. Med.1998;65(1):5-13). Using human bone marrow clonogenic assays, the CC₅₀values of mCyd in CFU-GM and BFU-E were 14.1±4.5 and 13.9±3.2 μM (seeTable 5). mCyd was significantly less toxic to bone marrow cells thanboth ZDV and RBV (Table 5). TABLE 5 Bone Marrow Toxicity of mCyd inGranulocyte Macrophage Progenitor and Erythrocyte Precursor CellsCFU-GM^(a) BFU-E^(a) Compound CC₅₀ (μM) CC₅₀ (μM) mCyd 14.1 ± 4.5 μM13.9 ± 3.2 ZDV 0.89 ± 0.47 0.35 ± 0.28 RBV 7.49 ± 2.20 0.99 ± 0.24

[0252] Effect on Mitochondrial Function

[0253] Antiviral nucleoside analogs approved for HIV therapy such asZDV, stavudine (d4T), didanosine (ddI), and zalcitabine (ddC) have beenoccasionally associated with clinically limiting delayed toxicities suchas peripheral neuropathy, myopathy, and pancreatitis (Browne M J, et al.J. Infect. Dis. 1993;167(1):21-29; Fischl M A, et al. Ann. Intern. Med.1993;18(10):762-769; Richman D D, et al. N. Engl. J. Med. 1987;317(4):192-197; Yarchoan R, et al. Lancet 1990;336(8714):526-529). Theseclinical adverse events have been attributed by some experts toinhibition of mitochondrial function due to reduction in mitochondrialDNA (mtDNA) content and nucleoside analog incorporation into mtDNA. Inaddition, one particular nucleoside analog, fialuridine(1,-2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl-5-iodo-uracil;FIAU)—caused hepatic failure, pancreatitis, neuropathy, myopathy andlactic acidosis due to direct mitochondrial toxicity (McKenzie R, et al.N Engl J Med 1995;333(17):1099-1105). Drug-associated increases inlactic acid production can be considered a marker of impairedmitochondrial function or oxidative phosphorylation. (Colacino, J. M.Antiviral Res 1996 29(2-3): 125-39).

[0254] To assess the potential of mCyd to produce mitochondrialtoxicity, several in vitro studies were conducted using the humanhepatoma cell lines HepG2 or Huh7. These studies included analysis oflactic acid production, mtDNA content, and determination of changes inmorphology (e.g., loss of cristae, matrix dissolution and swelling, andlipid droplet formation) of mitochondrial ultrastructure.

[0255] The effects of mCyd on mitochondria are presented in Table 6. Nodifferences were observed in lactic acid production in mCyd-treatedcells versus untreated cells at up to 50 μM mCyd in Huh7 cells or 10 μMmCyd in HepG2 cells. A modest (38%) increase in lactic acid productionwas seen in HepG2 cells treated with 50 μM mCyd. The significance ofthis finding is unclear, particularly since mCyd is unlikely to attain aplasma concentration of 50 μM in the clinic. For comparison, lactic acidproduction increases by 100% over control cells in cells treated with 10μM FIAU (Cui L, Yoon, et al. J. Clin. Invest. 1995;95:555-563). Exposureof HepG2 cells to mCyd for 6 or 14 days at concentrations up to 50 μMhad no negative effect on mitochondrial DNA content compared to a 56 or80% reduction in ddC-treated cells, respectively.

[0256] Following 14 days of exposure to 10 μM mCyd, the ultrastructureof HepG2 cells, and in particular mitochondria, was examined bytransmission electron microscopy. No changes in cell architecture, or inmitochondrial number or morphology (including cristae), were observed inthe majority of cells. In 17% of the cells, 1 to 2 mitochondria out ofan average of 25 per cell appeared enlarged. Such minor changes would beunlikely to have any significant impact on mitochondrial function.ddC-treated cells showed abnormal mitochondrial morphology with loss ofcristae, and the accumulation of fat droplets. (Medina, D. J., C. H.Tsai, et al. Antimicrob. Agents Chemother. 1994 38(8): 1824-8; Lewis W,et al. J. Clin. Invest. 1992;89(4):1354-1360., Lewis, L. D., F. M.Hamzeh, et al. Antimicrob. Agents Chemother. 1992 36(9): 2061-5). TABLE6 Effect of mCyd on Hepatocyte Proliferation, Mitochondrial Function,and Morphology in HepG2 Cells Electron L-Lactate mtDNA/nuclear DNAMicroscopy^(c) (% of Control^(a)) (% of Control^(b)) Lipid Conc HepG2Huh7 6 day 14 day Droplet Mito. Agent (μM) Cells Cells TreatmentTreatment Form. Morphol. Cont. 0 100 100 100 100 Negative Normal mCyd 10 98.6 ± 7.3 98.0 ± 12.3 117.3 ± 17.5 99.7 ± 23.9 Negative Normal^(d) 50138.0 ± 8.9 97.1 ± 10.1 158.2 ± 17.5 83.0 ± 15.5 nd nd ddC 1 nd nd  44.3± 9.3 19.6 ± 8.2 nd nd 10 nd nd nd nd Positive Loss of Cristae

[0257] Effect on Human DNA Polymerases α, β, and γ

[0258] The cellular DNA polymerases are responsible for normal nuclearand mitochondrial DNA synthesis and repair. Nucleoside analogtriphosphates are potential inhibitors of DNA polymerases and hencecould disrupt critical cell functions. In particular, the inhibition ofhuman polymerase γ, the enzyme responsible for mitochondrial DNAsynthesis, has been linked to defects in mitochondrial function (Lewis,W., E. S. Levine, et al. Proceedings of the National Academy ofSciences, USA 1996 93(8): 3592-7). Experiments were undertaken todetermine if mCyd-TP inhibited human DNA polymerases. As shown in Table7 mCyd-TP was not a substrate for human DNA polymerases α, β, or γ. Even1 mM mCyd-TP failed to inhibit these enzymes by 50% in the majority ofreplicate assays and IC₅₀ values could only be determined to be inexcess of 880-1000 μM. In contrast, ddC was a potent inhibitor of allthree human DNA polymerases and of polymerases β and γ in particular(IC₅₀s of 4.8 and 2.7 μM, respectively). Potent inhibition was also seenfor the control drug, actinomycin D, a known inhibitor ofDNA-dependent-DNA polymerases. TABLE 7 Inhibition of Human Polymerasesby mCyd-Triphosphate IC₅₀ (μM) mCyd-TP^(a) ddC-TP^(b) Act. D^(a) Polα >1000   78 ± 23.4 5.8 ± 3.1 Pol β ≧883.3 ± 165 4.8 ± 1   7.9 ± 3   Polγ ≧929.3 ± 100 2.7 ± 1   15.5 ± 4  

EXAMPLE 5 In Vitro Antiviral Activity Against BVDV

[0259] Compounds can exhibit anti-flavivirus or pestivirus activity byinhibiting flavivirus or pestivirus polymerase, by inhibiting otherenzymes needed in the replication cycle, or by other pathways.

[0260] Plaque Reduction Assay

[0261] For each compound the effective concentration was determined induplicate 24-well plates by plaque reduction assays. Cell monolayerswere infected with 100 PFU/well of virus. Then, serial dilutions of testcompounds in MEM supplemented with 2% inactivated serum and 0.75% ofmethyl cellulose were added to the monolayers. Cultures were furtherincubated at 37° C. for 3 days, then fixed with 50% ethanol and 0.8%Crystal Violet, washed and air-dried. Then plaques were counted todetermine the concentration to obtain 90% virus suppression.

[0262] Yield Reduction Assay

[0263] For each compound the concentration to obtain a 6-log reductionin viral load was determined in duplicate 24-well plates by yieldreduction assays. The assay was performed as described by Baginski, S.G.; Pevear, D. C.; Seipel, M.; Sun, S. C. C.; Benetatos, C. A.;Chunduru, S. K.; Rice, C. M. and M. S. Collett “Mechanism of action of apestivirus antiviral compound” PNAS USA 2000, 97(14), 7981-7986, withminor modifications. Briefly, MDBK cells were seeded onto 24-well plates(2×105 cells per well) 24 hours before infection with BVDV (NADL strain)at a multiplicity of infection (MOI) of 0.1 PFU per cell. Serialdilutions of test compounds were added to cells in a final concentrationof 0.5% DMSO in growth medium. Each dilution was tested in triplicate.After three days, cell cultures (cell monolayers and supernatants) werelysed by three freeze-thaw cycles, and virus yield was quantified byplaque assay. Briefly, MDBK cells were seeded onto 6-well plates (5×10⁵cells per well) 24 h before use. Cells were inoculated with 0.2 mL oftest lysates for 1 hour, washed and overlaid with 0.5% agarose in growthmedium. After 3 days, cell monolayers were fixed with 3.5% formaldehydeand stained with 1% crystal violet (w/v in 50% ethanol) to visualizeplaques. The plaques were counted to determine the concentration toobtain a 6-log reduction in viral load.

[0264] Studies on the antiviral activity of mCyd in cultured cells wereconducted. The primary assay used to determine mCyd antiviral potencywas a BVDV-based cell-protection assay (CPA). This assay measures theability of mCyd to protect growing MDBK bovine kidney cells fromdestruction by a cytopathic NADL strain of BVDV. The cytotoxicity of thetest drug on uninfected cells was measured in parallel. The antiviralactivities of mCyd and ribavirin in the CPA are compared in Table 8a.mCyd effectively protected de novo-infected MDBK cells in aconcentration-dependent manner with an EC₅₀=0.67±0.22 μM (Table 8a).mCyd afforded complete cytoprotection at concentrations well below theCC₅₀ for mCyd in this assay (38±9 μM). In the CPA, as well as in otherassays described below, ribavirin showed no clear antiviral effect:significant (50% or more) cell protection was not achieved in mostassays as the cytotoxicity of ribavirin overlapped and masked theprotective effect. Thus, ribavirin gave a CC₅₀ of 4.3±0.6 μM and anEC₅₀>4.3 μM in the CPA. TABLE 8a In Vitro Activity of mCyd Against BVDVin the Cell Protection Assay Compound n EC₅₀, μM CC₅₀, μM mCyd 11 0.67 ±0.22 38 ± 9  RBV  3 >4.3 4.3 ± 0.6

[0265] TABLE 8b CC₅₀ Test Results for β-D-2′-C-methyl-cytidine (CompoundG), 3′-O-valinyl ester of β-D-2′-C-methyl-cytidine dihydrochloride salt(Compound M), and β-D-2′-C-methyl-uracil (Compound N) Com- DENV poundCC₅₀ BVDV YFV 2 WNV CVB-2 Sb-1 REO G 34 2.3 54 95 80 12 11.5 13 M 24 5.882 >100 82 12 14 22 N >100 18 100 > or 80 >100 55 >100 =100

[0266] TABLE 8c CC₅₀ and EC₅₀ Test Results for β-D-2′-C-methyl-cytidine(Compound G) CC₅₀ CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4Vero 76 BHK Sb-1 CVB-2 CVB-3 CVB-4 CVA-9 REO-1 G 34 >100 >100 6 11 9 1326 13

[0267] The overall antiviral potency of mCyd was determined againstdifferent strains of BVDV and both cytopathic (cp) and noncytopatbic(ncp) biotypes in cell protection assays as well as in plaque reductionand yield reduction assays. The latter assays measure the output ofinfectious virus from cells and hence provide a stringent test ofantiviral efficacy. The different data sets from all three assays showagreement as summarized in Table 9. The range of 50% and 90% effectiveinhibitory concentration (EC₅₀ and EC₉₀) values for mCyd was 0.3 to 2.8μM and 0.87 to 4.8 μM, respectively.

[0268] In the BVDV yield reduction assay, subcytotoxic concentrations(circa 20 μM) of mCyd suppressed de novo BVDV production by up to 6log₁₀, to the point where no infectious virus was detected. A 4 log₁₀effective reduction in BVDV production (EC_(4 log 10) or EC_(99.99)) wasattained between 6.0 and 13.9 μM mCyd. In contrast, interferon alpha 2b(IFN α2b), although active against BVDV in this assay (EC₅₀ 2.6 IU perml), never gave more than 2 logs of viral reduction, even at 1000 IU perml.

[0269] Thus, the antiviral effect of mCyd against BVDV was much greaterthan that of IFNα2b or RBV.

EXAMPLE 6 In Vitro Antiviral Activity Against Other Positive-Strand RNAViruses

[0270] mCyd has been tested for efficacy against positive-strand RNAviruses other than BVDV. Data obtained are summarized in Table 9 and 10.Against flaviruses, mCyd showed modest activity. The composite EC₅₀ranges (in μM) determined from both sites were: West Nile virus (46-97);Yellow Fever virus (9-80); and Dengue virus (59-95). For mCyd againstthe alpha virus, Venezuelan equine encephalitis virus, EC₅₀ values were1.3-45 μM. mCyd was broadly active against picornaviruses, such as poliovirus (EC₅₀=6 μM), coxsackie virus (EC₅₀=15 μM), rhinovirus types 5 and14 (EC₅₀s=<0.1 and 0.6 μg/ml) and rhinovirus type 2 (EC₅₀ 2-10 μM). mCydwas generally inactive against all RNA and DNA viruses tested except forthe positive-strand RNA viruses. mCyd was also found to have no activityagainst HIV in MT-4 human T lymphocyte cells or HBV in HepG2.2.15 cells.TABLE 9 In Vitro Antiviral Activity of mCyd Against Plus-Strand RNAViruses Method of Virus Cell Antiviral Efficacy (μM) Assay Type Type nEC₅₀ EC₉₀ EC_(4 log) Cell BVDV MDBK 11 0.67 ± 0.22 Protection NADL cpAssay Yield BVDV MDBK 3 2.77 ± 1.16  4.8 ± 1.55 13.9 ± 3.07 ReductionNADL cp Assay BVDV MDBK 6 0.30 ± 0.07 0.87 ± 0.18 6.03 ± 1.41 New York-1ncp BVDV MDBK 1 0.68 1.73 8.22 I-NADL cp BVDV MDBK 1 0.59 1.49 7.14I-N-dIns ncp Plaque BVDV MDBK 3 2.57 ± 0.35 4.63 ± 0.72 Reduction NADLcp Assay Cell West Nile Virus BHK 3 63-97 Protection Assay Cell YellowFever BHK 1 60-80 Protection Virus 17D Assay DENV-2 BHK 2 95 Cell DENV-4BHK 1 59 Protection Assay Polio Virus Plaque Sb-1 VERO 1 6 ReductionAssay Plaque Coxsackie VERO 1 15 Reduction Virus B2 Assay

[0271] TABLE 10 In Vitro Antiviral Activity, Selectivity, andCytotoxicity of mCyd Virus (Cell line)^(a) EC₅₀ ^(b) (μM) CC₅₀ ^(c) (μM)WNV (Vero) 46 114-124 YFV (Vero)  9-30 150->200 VEE (Vero) 1.3-45  >200HSV-1 (HFF)^(d) >100 >100 HSV-2 (HFF)^(d) >100 >100 VZV (HFF)^(d) >2067.8 EBV (Daudi)^(d) 25.5 >50 HCMV (HFF)^(d)  9.9-15.6 67-73 MCMV(MEF) >0.8 2.4 Influenza A/H1N1 (MDCK) >200 >200 Influenza A/H3N2(MDCK) >20 45-65 Influenza B (MDCK) >200  55-140 Adenovirus type 1(A549) >200 >200 Parainfluenza type 3 (MA-104) >200 >200 Rhinovirus type2 (KB)  2-10 >200 Rhinovirus type 5 (KB)^(d) 0.6 20-30 Rhinovirus type14 (HeLa-Ohio)^(d) <0.1  20->100 RSV type A (MA-104) >200 200 Punta ToroA (LLC-MK2) >200 >200

EXAMPLE 7 Multiplicity of Infection (MOI) and Antiviral Efficacy

[0272] The cell protection assay format was used to test the effect ofincreasing the amount of BVDV virus on the EC₅₀ of mCyd. Increasing themultiplicity of infection (MOI) of BVDV in this assay from 0.04 to 0.16,caused the EC₅₀ of mCyd to increase linearly from 0.5 μM toapproximately 2.2 μM.

EXAMPLE 8 Viral Rebound in mCyd Treated Cells

[0273] The effect of discontinuing treatment with mCyd was tested inMDBK cells persistently infected with a noncytopathic strain (strainI-N-dIns) of BVDV. Upon passaging in cell culture, these cellscontinuously produce anywhere from 10⁶ to >10⁷ infectious virusparticles per ml of media. This virus can be measured by adding culturesupernatants from treated MDBK (BVDV) cells to uninfected MDBK cells andcounting the number of resultant viral foci after disclosure byimmunostaining with a BVDV-specific antibody. Treatment of apersistently infected cell line with 4 μM mCyd for one cell passage (3days) reduced the BVDV titer by approximately 3 log₁₀ from pretreatmentand control cell levels of just under 10⁷ infectious units per ml. Atthis point, mCyd treatment was discontinued. Within a single passage,BVDV titers rebounded to untreated control levels of just over 107infectious units per ml.

EXAMPLE 9 Mechanism of Action

[0274] In standard BVDV CPA assays, mCyd treatment results in a markedincrease in total cellular RNA content as cells grow, protected from thecytopathic effects of BVDV. This is coupled with a marked decrease inthe production of BVDV RNA due to mCyd. Conversely, in the absence ofmCyd, total cellular RNA actually decreases as BVDV RNA rises due to thedestruction of the cells by the cytopathic virus. To further test theeffect of mCyd on viral and cellular RNAs, the accumulation ofintracellular BVDV RNA was monitored in MDBK cells 18-hours postinfection (after approximately one cycle of virus replication) usingReal Time RT-PCR. In parallel, a cellular housekeeping ribosomal proteinmRNA (rig S15 mRNA) was also quantitated by RT-PCR using specificprimers. The results showed that mCyd dramatically reduced BVDV RNAlevels in de novo-infected MDBK cells with an EC₅₀ of 1.7 μM and an EC₉₀of 2.3 μM. The maximum viral RNA reduction was 4 log₁₀ at the highestinhibitor concentration tested (125 μM). No effect on the level of therig S15 cellular mRNA control was observed. Together, the precedingfindings suggest that mCyd inhibited BVDV by specifically interferingwith viral genome RNA synthesis without impacting cellular RNA content.This idea is further supported by the observation (Table 4a) thatinhibition of RNA synthesis as measured by [³H]-uridine uptake in HepG2cells requires high concentrations of mCyd (EC₅₀=186 μM).

[0275] In in vitro studies using purified BVDV NS5B RNA-dependent RNApolymerase (Kao, C. C., A. M. Del Vecchio, et al. (1999). “De novoinitiation of RNA synthesis by a recombinant flaviviridae RNA-dependentRNA polymerase.” Virology 253(1): 1-7) and synthetic RNA templates,mCyd-TP inhibited RNA synthesis with an IC₅₀ of 0.74 μM and was acompetitive inhibitor of BVDV NS5B RNA-dependent RNA polymerase withrespect to the natural CTP substrate. The inhibition constant (K_(i))for mCyd-TP was 0.16 μM and the Michaelis-Menten constant (K_(m)) forCTP was 0.03 μM. Inhibition of RNA synthesis by mCyd-TP required thepresence of a cognate G residue in the RNA template. The effect ofmCyd-TP on RNA synthesis in the absence of CTP was investigated in moredetail using a series of short (21mer) synthetic RNA templatescontaining a single G residue, which was moved progressively along thetemplate. Analysis of the newly synthesized transcripts generated fromthese templates in the presence of mCyd-TP revealed that RNA elongationcontinued only as far as the G residue, then stopped (FIG. 2). Intemplates containing more than one G residue, RNA synthesis stopped atthe first G residue encountered by the polymerase. These data stronglysuggest that m-Cyd-TP is acting as a non-obligate chain terminator. Themechanism of this apparent chain termination is under furtherinvestigation.

EXAMPLE 10 Eradication of a Persistent BVDV Infection

[0276] The ability of mCyd to eradicate a viral infection was tested inMDBK cells persistently infected with a noncytopathic strain of BVDV(strain I-N-dIns). (Vassilev, V. B. and R. O. Donis Virus Res 200069(2): 95-107.) Compared to untreated cells, treatment of persistentlyinfected cells with 16 μM mCyd reduced virus production from more than 6logs of virus per ml to undetectable levels within two cell passages (3to 4 days per passage). No further virus production was seen uponcontinued treatment with mCyd through passage 12. At passages 8, 9 and10 (arrows, FIG. 3), a portion of cells was cultured for two furtherpassages in the absence of drug to give enough time for mCyd-TP to decayand virus replication to resume. The culture media from the cells wererepeatedly tested for the re-emergence of virus by adding culturesupernatants from treated MDBK (BVDV) cells to uninfected MDBK cells andcounting the resultant viral foci after disclosure by immunostainingwith a BVDV-specific antibody. Although this assay can detect a singlevirus particle, no virus emerged from the cells post drug treatment.Thus, treatment with mCyd for 8 or more passages was sufficient toeliminate virus from the persistently infected cells.

EXAMPLE 11 Combination Studies with Interferon Alpha 2B

[0277] The first study, performed in MDBK cells persistently infectedwith the New York-1 (NY-1) strain of BVDV, compared the effect ofmonotherapy with either mCyd (8 μM) or interferon alpha 2b (200 IU/ml),or the two drugs in combination (FIG. 4A). In this experiment, 8 μM mCydalone reduced viral titers by approximately 3.5 log₁₀ after one passageto a level that was maintained for two more passages. Interferon alpha2b alone was essentially inactive against persistent BVDV infection(approximately 0.1 log₁₀ reduction in virus titer) despite being activeagainst de novo BVDV infection. However, the combination of mCyd plusinterferon alpha 2b reduced virus to undetectable levels by the secondpassage and clearly showed better efficacy to either monotherapy.

[0278] In a follow up study (FIG. 4B) of MDBK cells persistentlyinfected with the I-N-dIns noncytopathic strain of BVDV, mCyd wassupplied at fixed doses of 0, 2, 4 and 8 μM, while interferon alpha 2bwas titrated from 0 to 2,000 IU per ml. Again, interferon alpha 2b wasessentially inactive (0.1 log reduction in viral titer), while mCydalone inhibited BVDV (strain I-N-dIns) propagation in a dose-dependentmanner. mCyd at 8 μM reduced virus production by 6.2 log₁₀, to almostbackground levels.

EXAMPLE 12 Resistance Development

[0279] In early cell culture studies, repeated passaging of a cytopathicstrain of BVDV in MDBK cells in the presence of mCyd failed to generateresistant mutants, suggesting that the isolation of mCyd-resistant BVDVmutants is difficult. However, studies in cell lines persistentlyinfected with noncytopathic forms of BVDV led to the selection ofresistant virus upon relatively prolonged treatment with mCyd atsuboptimal therapeutic concentrations of drug (2 to 8 μM, depending onthe experiment). In the representative experiment shown in FIG. 5A, thevirus was no longer detectable after two passages in the presence of 8μM mCyd, but re-emerged by passage 6. The lower titer of the re-emergentvirus is apparent from the data: resistant virus typically has a 10 foldor more lower titer than the wild-type virus and is easily suppressed byco-therapy with IntronA (FIG. 5A). The phenotype of the virus thatre-emerged was remarkably different from the initial wild-type virus: asshown in FIG. 5B, it yielded much smaller foci (typically, 3 to 10 timessmaller in diameter then those of the wild-type virus). This phenotypedid not change after prolonged passaging in culture in the presence ofthe inhibitor (at least 72 days), however, it quickly reverted to thewild-type phenotype (large foci) after the discontinuation of thetreatment.

[0280] RT-PCR sequencing of the resistant mutant was used to identifythe mutation responsible for resistance. Sequencing efforts were focusedon the NS5B RNA-dependent RNA polymerase region of BVDV, which wasassumed to be the likely target for a nucleoside inhibitor. A specificS405T amino-acid substitution was identified at the start of the highlyconserved B domain motif of the polymerase. The B domain is part of thepolymerase active site and is thought to be involved in nucleosidebinding (Lesburg, C. A., M. B. Cable, et al., Nature Structural Biology1999 6(10): 937-43). Resistance to nucleosides has been mapped to thisdomain for other viruses such as HBV (Ono et al, J Clin Invest. 2001February;107(4):449-55). To confirm that this mutation was responsiblefor the observed resistance, the mutation was reintroduced into thebackbone of a recombinant molecular clone of BVDV. The resulting clonewas indistinguishable in phenotypic properties from the isolated mutantvirus, confirming that the S405T mutation is responsible for resistanceand that the NS5B RNA-dependent RNA polymerase is the molecular targetfor mCyd. The highly conserved nature of this motif at the nucleotidesequence (Lai, V. C., C. C. Kao, et al. J Virol 1999 73(12): 10129-36)and structural level among positive-strand RNA viruses (including HCV)allows a prediction that the equivalent mutation in the HCV NS5BRNA-dependent RNA polymerase would likely be S282T.

[0281] S405T mutant BVDV was refractory to mCyd up to the highestconcentrations that could be tested (EC₅₀>32 μM), but was alsosignificantly impaired in viability compared to wild-type virus. Asnoted above, the S405T mutant exhibited a 1-2 log₁₀ lower titer thanwild-type BVDV and produced much smaller viral plaques. In addition, themutant virus showed a marked reduction in the rate of a single cycle ofreplication (>1000-fold lower virus titer at 12 h), and accumulated toabout 100 fold lower levels than the wild-type virus even after 36 h ofreplication (FIG. 5C). The virus also quickly reverted to wild-typevirus upon drug withdrawal. Finally, the mutant was also more sensitive(˜40 fold) to treatment with IFN alpha 2b than wild-type as shown inFIG. 5D.

[0282] A second, additional mutation, C446S, was observed upon furtherpassaging of the S405T mutant virus in the presence of drug. Thismutation occurs immediately prior to the essential GDD motif in the Cdomain of BVDV NS5B RNA-dependent RNA polymerase. Preliminary studiessuggest that a virus bearing both mutations does not replicatesignificantly better than the S405T mutant, hence the contribution ofthis mutation to viral fitness remains unclear.

EXAMPLE 13 In Vivo Antiviral Activity of val-mCyd in an Animal EfficacyModel

[0283] Chimpanzees chronically infected with HCV are the most widelyaccepted animal model of HCV infection in human patients (Lanford, R.E., C. Bigger, et al. Ilar J 2001 42(2): 117-26; Grakoui, A., H. L.Hanson, et al. Hepatology 2001 33(3): 489-95). A single in vivo study ofthe oral administration of val-mCyd in the chimpanzee model of chronichepatitis C virus infection has been conducted.

[0284] HCV genotyping on the five chimpanzees was performed by theSouthwest Foundation Primate Center as part of their mandated internalHealth and Maintenance Program, designed to ascertain the disease statusof all animals in the facility to identify potential safety hazards toemployees. The five chimpanzees used in this study exhibited a high HCVtiter in a genotyping R_(T) PCR assay that distinguishes genotype 1 HCVfrom all other genotypes, but does not distinguish genotype 1a from 1b.This indicated that the chimpanzees used in this study were infectedwith genotype 1 HCV (HCV-1). TABLE 11 Summary of Val-mCyd In VivoActivity Study in the Chimpanzee Model of Chronic HCV Infection StudySpecies Val-mCyd Doese Frequency/Route Description (N) (mg/kg) (n) ofAdministration Study Endpoints One-week antiviral Chimpanzee 10 and 20(2 each) QD × 7 days Serum HCV RNA, activity of mCyd in (5) [equivalentto 8.3 and (PO) serum chemistries, chronically hepatitis 16.6 mpk offree CBCs, general well C virus (genotype base], and vehicle being, andclinical 1)-infected control (1) observations chimpanzees

[0285] Seven-Day Antiviral Activity Study in the Chimpanzee Model ofChronic Hepatitis C Virus Infection

[0286] Four chimpanzees (2 animals per dose group at 10 mg/kg/day or 20mg/kg/day) received val-mCyd dihydrochloride, freshly dissolved in aflavorful fruit drink vehicle. These doses were equivalent to 8.3 and16.6 mg/kg/day of the val-mCyd free base, respectively. A fifth animaldosed with vehicle alone provided a placebo control. The study designincluded three pretreatment bleeds to establish the baseline fluctuationof viral load and three bleeds during the one week of treatment (on days2, 5 and 7 of therapy) to evaluate antiviral efficacy. The analysis wascompleted at the end of the one-week dosing period, with no furtherfollow up.

[0287] HCV RNA Determination

[0288] Serum levels of HCV RNA throughout the study were determinedindependently by two clinical hospital laboratories. HCV RNA was assayedusing a quantitative RT-PCR nucleic acid amplification test (RocheAmplicor HCV Monitor Test, version 2.0). This assay has a lower limit ofdetection (LLOD) of 600 IU/mL and a linear range of 600-850,000 IU/mL.

[0289] To aid in interpretation of the viral load declines seen duringtherapy, emphasis was placed on determining (i) the extent offluctuations in baseline HCV viral load in individual animals, and (ii)the inherent variability and reproducibility of the HCV viral loadassay. To address these issues, full viral load data sets obtained fromthe two laboratories were compared. The results from both sites werefound to be closely comparable and affirmed both the stability of thepretreatment HCV viral loads as well as the reliability of the HCV RocheAmplicor assay. To present the most balanced view of the study, the meanvalues derived by combining both data sets were used to generate theresults presented in FIGS. 6 and 7. FIG. 6 presents the averaged datafor dose cohorts, while FIG. 7 presents the individual animal data. Thechanges in viral load from baseline seen during therapy for each animalat each site are also summarized in Table 12.

[0290] The HCV viral load analysis from the two sites revealed thatpretreatment HCV viral loads were (i) very similar among all fiveanimals and all 3 dose groups, and (ii) very stable over the 3-weekpretreatment period. The mean pretreatment log₁₀ viral load and standarddeviations among the five individual animals were 5.8±0.1 (site 1) and5.6±0.1(site 2). These data indicated that the c.v. (coefficient ofvariance) of the assay is only around 2% at both sites. The largestfluctuation in HCV viral load seen in any animal during pretreatment wasapproximately 0.3 log₁₀.

[0291] As seen in FIGS. 6 and 7, once a day oral delivery of val-mCydproduced a rapid antiviral effect that was not seen for the placeboanimal, nor during the pretreatment period. Viral titers weresubstantially reduced from baseline after two days of therapy for allanimals receiving val-mCyd, and tended to fall further under continuedtherapy in the two treatment arms. By the end of treatment (day 7), themean reductions from baseline HCV viral load were 0.83 log₁₀ and 1.05log₁₀ for the 8.3 and 16.6 mg/kg/day dose groups, respectively. Thetiter of the placebo animal remained essentially unchanged from baselineduring the therapy period.

[0292] An analysis of the data from the two quantification sites on thechanges in baseline HCV viral load in response to therapy is presentedin Table 12. Overall, the two data sets agree well, confirming thereliability of the assay. With the exception of animal 501, thedifference in viral load between the two sites was generally 0.3 log₁₀or less, similar to the fluctuation observed during the pretreatmentperiod. For animal 501, the discrepancy was closer to 0.5 log₁₀. Theviral load drop seen in response to therapy varied from 0.436 (animal501, site 1) to 1.514 log₁₀ (animal 497, site 2). The latter correspondsto a change in HCV viral load from 535,000 (pretreatment) to 16,500 (day7) genomes per ml. TABLE 12 Summary of Changes in Baseline Log₁₀ HCV RNAViral Load During Therapy Dose (mpk) Animal ID Site Day 2 Day 5 Day 7 0499 1 −0.00041 −0.11518 0.14085 2 −0.06604 0.10612 −0.16273 8.3 500 1−1.15634 −0.40385 −0.80507 2 −1.07902 −0.55027 −1.06259 8.3 501 1−0.25180 −0.36179 −0.43610 2 −0.45201 −0.71254 −0.90034 16.6 497 1−0.72148 −0.90704 −1.27723 2 −0.85561 −1.01993 −1.51351 16.6 498 1−0.29472 −0.28139 −0.60304 2 −0.65846 −0.55966 −0.69138

[0293] Exposure of Chimpanzees to mCyd

[0294] Limited HPLC analyses were perfomed to determine theconcentration of mCyd attained in the sera of chimpanzees followingdosing with val-mCyd. In sera drawn 1 to 2 hours post dose on days 2 and5 of dosing, mCyd levels were typically between 2.9 and 12.1 μM (750 and3100 ng/mL, respectively) in treated animals. No mCyd was detected inpretreatment sera or in the placebo control sera. Within 24 hours of thefinal dose, serum levels of mCyd had fallen to 0.2 to 0.4 μM (50 and 100mg/mL, respectively). No mUrd was detected in any sera samples althoughthe methodology used has a lower limit of quantification of 0.4 μM (100ng/mL) for mUrd.

[0295] Safety of mCyd in the Chimpanzee Model of Chronic HCV Infection

[0296] Chimpanzees were monitored by trained veterinarians throughoutthe study for weight loss, temperature, appetite, and general wellbeing, as well as for blood chemistry profile and CBCs. No adverseevents due to drug were noted. The drug appeared to be well tolerated byall four treated animals. All five animals lost some weight during thestudy and showed some aspartate aminotransferase (AST) elevations, butthese are normal occurrences related to sedation procedures used, ratherthan study drug. A single animal experienced an alanine aminotransferase(ALT) flare in the pretreatment period prior to the start of dosing, butthe ALT levels diminished during treatment. Thus, this isolated ALTevent was not attributable to drug.

EXAMPLE 14 In Vitro Metabolism

[0297] Studies were conducted to determine the stability of val-mCyd andmCyd in human plasma. Val-mCyd was incubated in human plasma at 0, 21 or37° C. and samples analyzed at various time points up to 10 hours (FIG.8). At 37° C., val-mCyd was effectively converted to mCyd, with only 2%of the input val-mCyd remaining after 10 hours. The in vitro half-lifeof val-mCyd in human plasma at 37° C. was 1.81 hours. In studies of thein vitro stability of mCyd in human plasma, or upon treatment with acrude preparation enriched in human cytidine/deoxycytidine deaminaseenzymes, mCyd remained essentially unchanged and no deamination to theuridine derivative of mCyd (mUrd) occurred after incubation at 37° C.Only in rhesus and cynomologus monkey plasma was limited deaminationobserved. Incubation of mCyd at 37° C. in cynomologus monkey plasmayielded 6.7 and 13.0% of mUrd deamination product after 24 and 48 hours,respectively, under conditions where control cytidine analogs wereextensively deaminated.

[0298] In addition to the TP derivatives of mCyd and mUrd, minor amountsof mCyd-5′-diphosphate, mCyd-DP, roughly 10% the amount of thecorresponding TP, were seen in all three cell types. Lesser amounts ofmUrd-DP were detected only in two cell types, primary human hepatocytesand MDBK cells. No monophosphate (MP) metabolites were detected in anycell type. There was no trace of any intracellular mUrd and no evidencefor the formation of liponucleotide metabolites such as the5′-diphosphocholine species seen upon the cellular metabolism of othercytidine analogs.

[0299]FIG. 9 shows the decay profile of mCyd-TP determined followingexposure of HepG2 cells to 10 μM [³H]-mCyd for 24 hours. The apparentintracellular half-life of the mCyd-TP was 13.9±2.2 hours in HepG2 cellsand 7.6±0.6 hours in MDBK cells: the data were not suitable forcalculating the half life of mUrd-TP. The long half life of mCyd-TP inhuman hepatoma cells supported the notion of once-a-day dosing forval-mCyd in clinical trials for HCV therapy. Phosphorylation of mCydoccurred in a dose-dependent manner up to 50 μM drug in all three celltypes, as shown for HepG2 cells in FIG. 9C. Other than the specificdifferences noted above, the phosphorylation pattern detected in primaryhuman hepatocytes was qualitatively similar to that obtained using HepG2or MDBK cells.

[0300] Contribution of mUrd

[0301] In addition to the intracellular active moiety, mCyd-TP, cellsfrom different species have been shown to produce variable and lesseramounts of a second triphosphate, mUrd-TP, via deamination ofintracellular mCyd species. The activity of mUrd-TP against BVDV NS5BRNA-dependent RNA polymerase has not been tested to date but is planned.To date, data from exploratory cell culture studies on the antiviralefficacy and cytotoxicity of mUrd suggest that mUrd (a) is about 10-foldless potent than mCyd against BVDV; (b) has essentially no antiviralactivity against a wide spectrum of other viruses; and (c) is negativewhen tested at high concentrations in a variety of cytotoxicity tests(including bone marrow assays, mitochondrial function assays andincorporation into cellular nucleic acid). Based on these results, itappeared that the contribution of mUrd to the overall antiviral activityor cytotoxicity profile of mCyd is likely to be minor. Extensivetoxicology coverage for the mUrd metabolite of mCyd exists fromsubchronic studies conducted with val-mCyd in the monkey.

EXAMPLE 15 Cellular Pathways for Metabolic Activation

[0302] The nature of the enzyme responsible for the phosphorylation ofmCyd was investigated in substrate competition experiments. Cytidine(Cyd) is a natural substrate of cytosolic uridine-cytidine kinase (UCK),the pyrimidine salvage enzyme responsible for conversion of Cyd toCyd-5′-monophosphate (CMP). The intracellular phosphorylation of mCyd tomCyd-TP was reduced in the presence of cytidine or uridine in adose-dependent fashion with EC₅₀ values of 19.17±4.67 μM for cytidineand 20.92±7.10 μM for uridine. In contrast, deoxycytidine, a substratefor the enzyme deoxycytidine kinase (dCK), had little effect on theformation of mCyd-TP with an EC₅₀>100 μM. The inhibition of mCydphosphorylation by both cytidine and uridine, but not deoxycytidine,suggests that mCyd is phosphorylated by the pyrimidine salvage enzyme,uridine-cytidine kinase (Van Rompay, A. R., A. Norda, et al. MolPharmacol 2001 59(5): 1181-6). Further studies are required to confirmthe proposed role of this kinase in the activation of mCyd.

EXAMPLE 16 Pathways for the Cellular Biosynthesis of mURD-TP

[0303] As outlined above, mUrd-TP is a minor metabolite arising tovarying extents in cells from different species. mUrd does not originatevia extracellular deamination of mCyd since mUrd was not seen in thecell medium which also lacks any deamination activities. The cellularmetabolism data are consistent with the idea that mUrd-TP arises via thebiotransformation of intracellular mCyd species. Consideration of theknown ribonucleoside metabolic pathways suggests that the most likelyroutes involve deamination of one of two mCyd species by two distinctdeamination enzymes: either mCyd-MP by a cytidylate deaminase (such asdeoxycytidylate deaminase, dCMPD), or of mCyd by cytidine deaminase(CD). Further phosphorylation steps lead to mUrd-TP. These possibilitiesare under further investigation.

EXAMPLE 17 Clinical Evaluation of val-mCyd

[0304] Patients who met eligibility criteria were randomized into thestudy at Baseline (Day 1), the first day of study drug administration.Each dosing cohort was 12 patients, randomized in a 10:2 ratio totreatment with drug or matching placebo. Patients visited the studycenter for protocol evaluations on Days 1, 2, 4, 8, 11, and 15. AfterDay 15, study drug was stopped. Thereafter, patients attended follow-upvisits on Days 16, 17, 22, and 29. Pharmacokinetic sampling wasperformed on the first and last days of treatment (Day 1 and Day 15) onall patients, under fasting conditions.

[0305] The antiviral effect of val-mCyd was assessed by (i) theproportion of patients with a ≧1.0 log₁₀ decrease from baseline in HCVRNA level at Day 15, (ii) the time to a ≧1.0 log₁₀ decrease in serum HCVRNA level, (iii) the change in HCV RNA level from Day 1 to Day 15, (iv)the change in HCV RNA level from Day 1 to Day 29, (v) the proportion ofpatients who experience return to baseline in serum HCV RNA level by Day29, and (vi) the relationship of val-mCyd dose to HCV RNA change fromDay 1 to Day 15.

[0306] Clinical Pharmacokinetics of mCyd after Oral Administration ofEscalating Doses of Val-mCyd

[0307] Pharmacokinetics were evaluated over a period of 8 h after thefirst dose on day 1 and after the last dose on day 15, with 24-h troughlevels monitored on days 2, 4, 8, 11 and 16, and a 48-h trough on day17. Plasma concentrations of mCyd, mUrd and Val-mCyd were measured by aHPLC/MS/MS methodology with a lower limit of quantitation (LOQ) at 20ng/ml.

[0308] The pharmacokinetics of mCyd was analyzed using anon-compartmental approach. As presented in the tables below, theprincipal pharmacokinetic parameters were comparable on day 1 and day15, indicative of no plasma drug accumulation after repeated dosing. Theplasma exposure also appeared to be a linear function of dose. As shownin the tables below, principal pharmacokinetic parameters of drugexposure (Cmax and AUC) doubled as doses escalated from 50 to 100 mg.TABLE 13 Pharmacokinetic parameters of mCyd at 50 mg C_(max) T_(max)AUG_(0-inf) t_(1/2) Parameters (ng/ml) (h) (ng/ml × h) (h) Day 1 Mean428.1 2.5 3118.7 4.1 SD 175.5 1.1 1246.4 0.6 CV % 41.0 43.2 40.0 13.8Day 15 Mean 362.7 2.2 3168.4 4.6 SD 165.7 1.0 1714.8 1.3 CV % 45.7 46.954.1 28.6

[0309] TABLE 14 Pharmacokinetic parameters of mCyd at 100 mg C_(max)T_(max) AUC_(0-inf) t_(1/2) Parameters (nglml) (h) (ng/ml × h) (h) Day 1Mean 982.1 2.6 6901.7 4.4 SD 453.2 1.0 2445.7 1.1 CV % 46.1 36.2 35.425.2 Day 15 Mean 1054.7 2.0 7667.5 4.2 SD 181.0 0.0 1391.5 0.5 CV % 17.20.0 18.1 11.7

[0310] The mean day 1 and day 15 plasma kinetic profiles of mCyd at 50and 100 mg are depicted in the FIG. 10.

[0311] In summary, following oral administration of val-mCyd, the parentcompound mCyd was detectable in the plasma of HCV-infected subjects.mCyd exhibited linear plasma pharmacokinetics in these subjects acrossthe two dose levels thus far examined. There was no apparentaccumulation of mCyd in subjects' plasma following 15 days of dailydosing at the doses thus far examined.

[0312] Antiviral Activity of mCyd after Oral Administration ofEscalating Doses of Val-mCyd Starting at 50 mg/day for 15 Days inHCV-Infected Patients

[0313] Serum HCV RNA levels were determined with the use of the AmplicorHCV Monitor™ assay v2.0 (Roche Molecular Systems, Branchburg, N.J.,USA), which utilizes polymerase chain reaction (PCR) methods. The lowerlimit of quantification (LLOQ) with this assay was estimated to beapproximately 600 IU/mL and the upper limit of quantification (ULOQ)with this assay was estimated to be approximately 500,000 IU/mL.

[0314] Serum samples for HCV RNA were obtained at screening (Day 42 to−7) to determine eligibility for the study. The Screening serum HCV RNAvalues must be ≧5 log10 IU/mL by the Amplicor HBV Monitor™ assay at thecentral study laboratory.

[0315] During the study period, serum samples for HCV RNA were obtainedat Baseline (Day 1), and at every protocol-stipulated post-Baselinestudy visit (Days 2, 4, 8, 11, 15, 16, 17, 22, and 29). Serum samplesfor HCV RNA were also collected during protocol-stipulated follow-upvisits for patients prematurely discontinued from the study.

[0316] The antiviral activity associated with the first two cohorts (50and 100 mg per day) in the ongoing study is summarized in the followingtables and graphs. Although the duration of dosing was short (15 days)and the initial dose levels low, there were already apparent effects onthe levels of HCV RNA in the plasma of infected patients. TABLE 15Summary Statistics of HCV RNA in Log₁₀ Scale Day Treatment −1 1 2 4 8 1115 16 17 22 29 Placebo N 6 5 5 4 4 4 4 4 3 4 3 Median 6.45 6.25 6.256.52 6.42 6.28 6.58 6.51 6.64 6.35 6.61 Mean 6.45 6.28 6.40 6.48 6.366.34 6.54 6.52 6.50 6.40 6.40 StdErr 0.25 0.12 0.15 0.18 0.24 0.16 0.110.19 0.31 0.23 0.30 50 mg N 10 10 10 10 10 10 10 10 10 10 10 Median 6.816.69 6.58 6.55 6.56 6.46 6.57 6.45 6.54 6.73 6.67 Mean 6.72 6.72 6.606.56 6.62 6.47 6.57 6.57 6.54 6.64 6.71 StdErr 0.11 0.11 0.12 0.06 0.100.09 0.08 0.11 0.08 0.10 0.09 100 mg N 11 10 10 10 9 10 10 9 9 10 4Median 6.75 6.93 6.80 6.46 6.59 6.56 6.41 6.40 6.72 6.66 6.71 Mean 6.606.68 6.52 6.43 6.42 6.36 6.30 6.23 6.65 6.53 6.67 StdErr 0.16 0.24 0.230.21 0.24 0.22 0.22 0.23 0.16 0.18 0.17

[0317] TABLE 16 Summary Statistics of Change From Baseline (Day 1) inLog₁₀ HCV RNA Day Treatment 2 4 8 11 15 16 17 22 29 Placebo N 5 4 4 4 44 3 4 3 Median 0.17 0.21 0.15 0.08 0.31 0.21 0.27 0.17 0.09 Mean 0.120.22 0.10 0.08 0.28 0.25 0.15 0.14 0.09 StdErr 0.09 0.12 0.16 0.06 0.150.10 0.18 0.09 0.16 50 mg N 10 10 10 10 10 10 10 10 10 Median −0.07−0.13 −0.06 −0.26 −0.10 −0.13 −0.21 −0.09 −0.04 Mean −0.13 −0.16 −0.11−0.26 −0.15 −0.15 −0.18 −0.09 −0.01 StdErr 0.05 0.07 0.05 0.06 0.08 0.050.07 0.06 0.10 100 mg N 10 10 9 10 10 9 9 10 4 Median −0.12 −0.24 −0.20−0.28 −0.43 −0.49 −0.24 −0.19 −0.12 Mean −0.16 −0.25 −0.21 −0.32 −0.38−0.39 −0.18 −0.15 0.13 StdErr 0.07 0.10 0.16 0.13 0.12 0.14 0.15 0.130.28

[0318]FIG. 11 depicts the Median Change From Baseline in Log₁₀ HCV RNABy Visit

[0319] The present invention is described by way of illustration in thefollowing examples. It will be understood by one of ordinary skill inthe art that these examples are in no way limiting and that variationsof detail can be made without departing from the spirit and scope of thepresent invention.

We claim:
 1. A compound of Formula (I) or a pharmaceutically acceptablesalt thereof:


2. The compound of claim 1, wherein the pharmaceutically acceptable saltis a hydrochloride salt.
 3. The compound of claim 1, wherein thepharmaceutically acceptable salt is the dihydrochloride salt of Formula(II).


4. A pharmaceutical composition comprising an effective amount of thecompound of Formula (I) or a pharmaceutically acceptable salt thereof totreat a Flaviviridae infection, in a pharmaceutically acceptablecarrier.


5. The pharmaceutical composition of claim 4 wherein thepharmaceutically acceptable salt is a hydrochloride salt.
 6. Thepharmaceutical composition of claim 4 wherein the pharmaceuticallyacceptable salt is a dihydrochloride salt.
 7. The pharmaceuticalcomposition of claim 4, whrein the pharmaceutically acceptable carrieris suitable for oral delivery.
 8. The pharmaceutical composition ofclaim 4, further comprising a second antiviral agent.
 9. Thepharmaceutical composition of claim 8, wherein the second anti-viralagent is selected from the group consisting of an interferon, ribavirin,interleukin, NS3 protease inhibitor, cysteine protease inhibitor,phenanthrenequinone, thiazolidine derivative, thiazolidine, benzanilide,a helicase inhibitor, a polymerase inhibitor, nucleotide analogue,gliotoxin, cerulenin, antisense phosphorothioate oligodeoxynucleotides,inhibitors of IRES-dependent translation, and a ribozyme.
 10. Thepharmaceutical composition of claim 8, wherein the second agent is aninterferon.
 11. The pharmaceutical composition of claim 8, wherein thesecond agent is selected from the group consisting of pegylatedinterferon alpha 2a, interferon alphacon-1, natural interferon,albuferon, interferon beta-1a, omega interferon, interferon alpha,interferon gamma, interferon tau, interferon delta and interferongamma-1b.
 12. The pharmaceutical composition of claim 8, wherein thesecond agent is interferon alpha
 2. 13. The pharmaceutical compositionof claim 4, wherein the compound is in the form of a dosage unit. 14.The composition of claim 13, wherein the dosage unit contains 0.01 to 50mg of the compound.
 15. The composition of claim 13, wherein said dosageunit is a tablet or capsule.
 16. The composition of claim 4, wherein thecompound is in substantially pure form.
 17. The compound of claims 1-3,wherein the compound is at least 90% by weight of the β-D-isomer. 18.The compoound of claims 1-3, wherein the compound is at least 95% byweight of the β-D-isomer.
 19. A method of treating a host infected witha flavivirus or pestivirus, comprising administering to a host in needthereof an effective amount of a compound having the structure ofFormula (I) or a pharmaceutically acceptable salt or prodrug thereofoptionally in a pharmaceutically acceptable carrier.


20. The method according to claim 19, wherein the pharmaceuticallyacceptable salt is a hydrochloride salt.
 21. The method according toclaim 19, wherein the pharmaceutically acceptable salt is adihydrochloride salt.
 22. The method according to claim 19, furthercomprising administering the compound in a pharmaceutically acceptablecarrier, diluent or excipient.
 23. The method according to claim 19,wherein the compound is administered in combination or alternation witha second anti-viral agent.
 24. The method according to claim 23, whereinthe second antiviral agent is selected from the group consisting ofinterferon, ribavirin, interleukin, an NS3 protease inhibitor, cysteineprotease inhibitor, thiazolidine derivative, thiazolidine, benzanilide,phenan-threnequinone, a helicase inhibitor, a polymerase inhibitor, anucleotide analogue, gliotoxin, cerulenin, antisense phosphorothioateoligodeoxynucleotides, inhibitor of IRES-dependent translation, and aribozyme.
 25. The method according to claim 23, wherein the secondantiviral agent is an interferon.
 26. The method according to claim 19,wherein the second antiviral agent is selected from the group consistingof pegylated interferon alpha 2a, interferon alphacon-1, naturalinterferon, albuferon, interferon beta-1a, omega interferon, interferonalpha, interferon gamma, interferon tau, interferon delta and interferongamma-1b.
 27. The method of claim 26, wherein the second antiviral agentis interferon alpha
 2. 28. The method of claim 19, wherein the host is ahuman.
 29. The method of claim 19, wherein the compound is in the formof a dosage unit.
 30. The method of claim 29, wherein the dosage unitcontains 10 to 500 mg of the compound.
 31. The method of claim 29,wherein said dosage unit is a tablet or capsule.
 32. The method of claim19, wherein the pharmaceutically acceptable carrier is suitable for oralor intravenous delivery.
 33. The method of claim 19, wherein thecompound is in administered in substantially pure form.
 34. The methodof claim 19, wherein the compound is at least 90% by weight of theβ-D-isomer.
 35. The method of claim 19, wherein the compound is at least95% by weight of the β-D-isomer.
 36. The method of claim 19, wherein thevirus is HCV.
 37. A compound of Formula I or II, wherein the 5′-hydroxylgroup is replaced with a 5′-OR, wherein R is phosphate; a stabilizedphosphate prodrug; acyl; alkyl; sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted; a lipid; an amino acid; acarbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R is independently H or phosphate.
 38. Apharmaceutical composition that comprises the compound of Formula I orII in a pharmaceutically acceptable carrier, wherein the 5′-hydroxylgroup is replaced with a 5′-OR, wherein R is phosphate; a stabilizedphosphate prodrug; acyl; alkyl; sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and; benzyl, wherein thephenyl group is optionally substituted; a lipid, an amino acid; acarbohydrate; a peptide; a cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R is independently H or phosphate.
 39. Amethod for treating a host infected with an RNA-dependant RNA polymerasevirus, comprising administering an effective amount of the compound ofFormula I or II in a pharmaceutically acceptable carrier, wherein the5′-hydroxyl group is replaced with a 5′-OR, wherein R is hydrogen,phosphate; a stabilized phosphate prodrug; acyl; alkyl; sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl and;benzyl, wherein the phenyl group is optionally substituted; a lipid, anamino acid; a carbohydrate; a peptide; a cholesterol; or otherpharmaceutically acceptable leaving group which when administered invivo is capable of providing a compound wherein R is independently H orphosphate.
 40. The method of claim 39 wherein the virus is aFlaviviridae virus.
 41. The method of claim 39 or 40 wherein theFlaviviridae virus is hepatitis C.
 42. The method of claims 39, 40 or 41wherein the host is human.
 43. The compound of claim 1, wherein thepharmaceutically acceptable salt is selected from tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate, formate,fumarate, propionate, glycolate, lactate, pyruvate, oxalate, maleate,salicyate, sulfate, sulfonate, nitrate, bicarbonate, hydrobromate,hydrobromide, hydroiodide, carbonate, and phosphoric acid salts.
 44. Thecomposition of claim 4, wherein the pharmaceutically acceptable salt isselected from tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, andα-glycerophosphate, formate, fumarate, propionate, glycolate, lactate,pyruvate, oxalate, maleate, salicyate, sulfate, sulfonate, nitrate,bicarbonate, hydrobromate, hydrobromide, hydroiodide, carbonate, andphosphoric acid salts.
 45. The method of claim 19, wherein thepharmaceutically acceptable salt is selected from tosylate,methanesulfonate, acetate, citrate, malonate, tartarate, succinate,benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate, formate,fumarate, propionate, glycolate, lactate, pyruvate, oxalate, maleate,salicyate, sulfate, sulfonate, nitrate, bicarbonate, hydrobromate,hydrobromide, hydroiodide, carbonate, and phosphoric acid salts.